Ultrasound-Mediated Microbubble Destruction Increases Renal Interstitial Capillary Permeability in Early Diabetic Nephropathy Rats

Diabetic nephropathy (DN) is defined as persistent proteinuria corresponding to a urinary albumin excretion rate >300 μg/mg in the absence of other non-diabetic renal diseases. The aim of this study was to determine if ultrasound (US)-mediated microbubble (MB) destruction could increase renal interstitial capillary permeability in early DN rats. Diabetes was induced with streptozotocin. DN rats presented with mild micro-albuminuria 30 d after onset of diabetes. DN rats (N = 120) were divided into four groups that received Evans blue (EB) followed by: (i) no treatment (control group); (ii) continuous ultrasonic irradiation for 5 min (frequency = 7.00 MHz, mechanical index = 0.9, peak rarefactional pressure = 2.38 MPa: US group); (iii) microbubble injection (0.05 mL/kg: MB group); and (iv) both ultrasound and microbubble injection (US + MB group). Another 8 DN rats were subjected to ultrasound and microbubbles and then injected with EB after 24 h (recovery group). EB content, EB extravasation and E-selectin mRNA and protein expression significantly increased, and interstitial capillary walls became discontinuous in the US + MB group. Neither hemorrhage nor necrosis was observed on renal histology. Urine samples were collected 24 h post-treatment. There was no hematuria, and the urinary albumin excretion rate did not increase after ultrasound-microbubble interaction detected by urinalysis. EB content returned to the control group level after 24 h, as assessed for the recovery group. In conclusion, ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability in DN rats, and should be considered a therapy for enhancing drug and gene delivery to the kidney in the future.     

Microbubble Stability is a Major Determinant of the Efficiency of Ultrasound and Microbubble Mediated in vivo Gene Transfer

In the search for an efficient nonviral gene therapy approach for the treatment of genetic disorders of cardiac and skeletal muscle such as Duchenne muscular dystrophy, ultrasound in combination with contrast enhancing microbubbles has emerged as a promising tool for safe and site-specific enhancement of gene delivery. Indeed, microbubble-enhanced gene transfer (MBGT) has been investigated for a wide variety of target sites using both reporter and therapeutic genes. Although a range of different microbubbles have been used for MBGT studies, comparison of their efficiencies is difficult because microbubble concentration and the ultrasound settings used for the application vary considerably. Only two studies to date have attempted a direct comparison of commercially available microbubbles, and both concluded that not all microbubbles show the same efficiencies with MBGT. Thus far, the reason for this is unclear. Here, the efficiency of three commercially available microbubbles—Optison, SonoVue and Sonazoid—was analyzed to understand the microbubble properties that are important for their function as an effective enhancer for gene transfer in vivo. In this study, plasmid DNA or antisense oligonucleotides were delivered by systemic injection with MBGT, focused on the heart. Gene delivery to the heart with equalized concentrations of the three microbubbles showed that Optison and Sonazoid are more efficient in MBGT compared with SonoVue, which showed the weakest gene transfer to the myocardium. Investigations into the properties of these microbubbles showed that size and shell composition did not directly influence MBGT, whereas the microbubbles with increased stability in an ultrasound field showed better MBGT results than those degrading faster. Moreover, the microbubble concentration used for MBGT was also found to be an important factor influencing the efficiency of MBGT. In conclusion, the stability of a microbubble was shown to be a major influential factor for its performance in MBGT, as is the concentration of the microbubbles used. These findings emphasize the importance of detailed investigations into the properties of microbubbles to allow the production of a microbubble specifically designed for optimum performance with MBGT. (E-mail: d.wells@imperial.ac.uk)     

超声靶向微泡破坏技术介导自杀基因在恶性肿瘤中的研究进展

恶性肿瘤是全球发病率和死亡率的一个重要原因,严重威胁人类的健康。作为其常规的治疗方法,化疗、放疗和手术仍有局限性。自杀基因疗法为各种形式的恶性肿瘤提供了一种极具前景的治疗手段。超声靶向微泡破坏(UTMD)技术为自杀基因的传递赋予了新的方式。本文着重就 UTMD 介导的自杀基因转染在恶性肿瘤治疗中的最新进展进行总结并对其未来发展提出展望。     

超声微泡造影剂对心肌组织的生物学效应及介导VEGF基因转染大鼠心肌的实验研究

目的 探讨超声微泡造影剂对心肌组织的生物学效应及其介导VEGF基因转染大鼠心肌的有效性。方法 18只健康雄性Wistar大鼠，取3只采用超声波在鼠胸壁破坏微泡造影剂，观察对心肌组织显微结构的影响。将另15只急性心肌梗死3天后的雄性Wistar大鼠分为3组，每组5只。第一组采用超声破坏微泡造影剂的方式，将pcDzVEGFm基因转染大鼠心肌至造影剂不再显影(约6min)；第二组尾静脉输入同等剂量携pcD。VEGF。基因的造影剂；第三组为对照。2周后，取缺血心肌组织行VEGF免疫组织化学染色，观察心肌组织血管内皮生长因子(VEGF)蛋白表达情况。结果超声波破坏微泡造影剂能使心肌组织充血，产生大量空泡，并有部分心肌细胞坏死。采用超声微泡造影剂介导的VEGF基因转染，能明显增强大鼠心肌组织VEGF蛋白的表达。结论 超声微泡造影剂能明显增强对组织的空化效应，其介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法。     

Ultrasound targeted microbubble destruction increases capillary permeability in hepatomas

Ultrasound-targeted microbubble destruction (UTMD) has evolved as a promising tool for organ-specific gene and drug delivery. Taking advantage of high local concentrations of therapeutic substances and transiently increased capillary permeability, UTMD could be used for the treatment of ultrasound accessible tumors. The aim of this study was to evaluate if UTMD can locally increase capillary permeability in a hepatoma model of the rat. Furthermore, we evaluated whether UTMD can transfect DNA into such tumors. Subcutaneous Morris hepatomas were induced in both hind limbs of ACI rats by cell injection. A total of 18 rats were divided into three groups. Only one tumor per rat was treated by ultrasound. The first group received injection of Evans blue, followed by UTMD. The second group received a phosphate-buffered saline solution infusion and ultrasound to the target tumor after Evans blue injection. The third group received UTMD first, followed by Evans blue injection. Tumors and control organs were harvested, and Evans blue extravasation was quantified. Another 12 rats received DNA-loaded microbubbles by UTMD to one tumor, encoding for luciferase. Evans blue injection followed by UTMD showed about fivefold higher Evans blue amount in the target tumors compared with the control tumors. In contrast, no significant difference in Evans blue content was detected between target and control tumors when ultrasound was applied without microbubbles or when UTMD was performed before Evans blue injection. Plasmid transfection was not successful. In conclusion, ultrasound targeted microbubble destruction is able to transiently increase capillary permeability in hepatomas. Using naked DNA, this technique does not seem to be feasible for noninvasive transfection of hepatomas.     

Ultrasound Imaging of Microbubble Activity during Sonoporation Pulse Sequences

Ultrasound-mediated drug delivery using the mechanical action of oscillating and/or collapsing microbubbles has been studied on many different experimental platforms, both in vitro and in vivo; however, the mechanisms remain to be elucidated. Many groups use sterile, enclosed chambers, such as Opticells and Clinicells, to optimize acoustic parameters in vitro needed for effective drug delivery in vivo, as well as for mechanistic investigation of sonoporation or the use of sound to permeate cell membranes. In these containers, cell monolayers are seeded on one side, and the remainder of the volume is filled with a solution containing microbubbles and a model drug. Ultrasound is then applied to study the effect of different parameters on model drug uptake in cell monolayers. Despite the simplicity of this system, the field has been unable to appropriately address what parameters and microbubble concentrations are most effective at enhancing drug uptake and minimizing cellular toxicity. In this work, a common in vitro sonoporation experimental setup was characterized through quantitative analysis of microbubble-dependent acoustic attenuation in combination with high-frame-rate and high-resolution imaging of bubble activity during sonoporation pulse sequences. The goal was to visualize the effect that ultrasound parameters have on microbubble activity. It was observed that under literature-derived sonoporation conditions (0.1–1 MPa, 20–1000 cycles and 10,000 to 10,000,000 microbubbles/mL), there is strong and non-linear acoustic attenuation, as well as bubble destruction, gas diffusion and bubble motion resulting in spatiotemporal pressure and concentration gradients. Ultimately, it was found that the acoustic conditions in common in vitro sonoporation setups are much more complex and confounding than often assumed.     

原子力声显微镜评价超声靶向微泡破坏法对大鼠心肌母细胞的影响

目的 探讨运用原子力声显微镜评价超声靶向微泡破坏法对大鼠心肌母细胞(H9C2)影响的可行性.方法 采用不同超声参数对H9C2细胞进行辐照处理.使用原子力声显微镜观察细胞骨架、形态结构的改变;采用台盼蓝染色法及荧光物质转入法检测细胞存活率及细胞膜通透性变化.结果 (1)通过比较辐照后H9C2细胞生存率及发光率变化,适宜参数为:频率1 MHz,声强1.0 W/cm2,占空比20％,辐照时间60 s;(2)原子力声显微镜可以完整显示H9C2细胞的形态结构,以形态像、声学像及三维像的差异区分细胞损伤的程度.结论 原子力声显微镜能够准确评估不同超声辐照参数对H9C2细胞的影响,优化超声靶向微泡破坏法转染参数.     

超声联合微泡辅助卡铂抑制A549细胞活性的适宜剂量研究

目的 探讨超声辅助卡铂抑制体外A549细胞活性的适宜卡铂剂量及超声能量参数。方法 96孔板接种A549细胞,加入浓度梯度0-400>μg/ml的卡铂培养24小时后,每孔加入 10μlCCK8 溶液,孵育 1.5 小时,酶标仪测定在450nm处的吸光度值,计算细胞抑制率,应用Graphpad Prism 5软件的“药物剂量-效应模型”计算卡铂24小时IC50。设置空白组、US组、USMB组、CBP组、CBP+US组、CBP+USMB组,卡铂浓度为50 μg/ml,声强梯度范围为0.2-2.2W/cm2,频率1MHz,占空比10%,辐照时间1分钟,培养24小时,CCK8法测细胞存活率,应用SPSS24.0分析存活率差异,筛选有效剂量。光学倒置显微镜观察各组细胞形态学变化。结果 卡铂24小时IC50为65.72μg/ml。抑制A549细胞活性的强弱排序为CBP+USMB组>CBP+US组>CBP组>USMB组>US组,CBP+USMB组与其余4组之间差异有统计学意义(P<0.001),CBP(50μg/ml)+USMB(0.2-1.0W/cm2)5组之间差异无统计学意义(P>0.05)。处理组均可见细胞形态不规则、贴壁性降低,以CBP+USMB组为著。结论 卡铂50μg/ml、声强0.6W/cm2为24小时联合疗法适宜剂量。超声联合微泡可以与卡铂协同抑制A549细胞活性,成为肿瘤联合疗法的新策略。     

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。     

Investigation Into the Impact of Diagnostic Ultrasound with Microbubbles on the Capillary Permeability of Rat Hepatomas

Ultrasound-targeted microbubble destruction (UTMD) takes advantage of transiently increased capillary permeability to enhance the release of tumor-specific drugs from blood vessels into sonicated tumor tissues. However, the application of focused ultrasound is limited because of the lack of an appropriate image-monitoring system. In this study, hepatoma-bearing Sprague-Dawley rats were insonicated with low-frequency diagnostic ultrasound and injected with Evans Blue (EB) dye and microbubbles through their tail veins to test changes in capillary permeability. We studied how the mechanical index, sonication duration and the injected microbubble (MB) concentration affect the hepatoma vascular permeability by quantitatively evaluating the EB delivery efficiency. Confocal laser scanning microscopy was used to observe the deposition of red fluorescence–dyed EB in tumor tissues. In addition, P-selectin, a type of biochemical marker that reflects vascular endothelial cell activation, was identified using an immunoblotting analysis. The experimental results reveal that EB delivery efficiency in tumor tissues was greater in groups with the diagnostic ultrasound–mediated UTMD (8.40 ± 0.71 %ID/g) than in groups without UTMD (1.73 ± 0.19 %ID/g) and EB delivery efficiency could be affected by MI, sonication duration and MB dose. The immunoblotting analysis indicates that diagnostic ultrasound–induced UTMD results in the vascular endothelial cell activation to increase capillary permeability, justifying the high quantity of EB deposited in tumor tissues.     

携galectin-7-siRNA 超声纳泡靶向治疗大鼠心脏移植急性排斥反应的实验研究

目的采用超声靶向微泡破坏技术介导galectin-7-siRNA转染,抑制大鼠移植心脏galectin-7表达,以达到靶向治疗心脏移植后急性排斥反应的目的。方法制备携galectin-7-siRNA的ICAM-1靶向纳泡;构建大鼠同系移植(ISO)及异系移植(ALLO)心脏模型,并分为4组:ISO+磷酸盐缓冲液(PBS)组、ALLO+PBS+低功率聚焦超声(LIFU)组、ALLO+微泡(NBs)组及ALLO+NBs+LIFU组。于移植后第1、3、5、7天分别行超声靶向微泡定位释放治疗,治疗后取材比较各组心脏galectin-7表达及急性排斥反应分级。结果制备的纳泡平均粒径(221.25±34.21)nm,平均电位(51.32±2.21)m V;galectin-7-siRNA携带率71.0%。ALLO+NBs+LIFU组galectin-7蛋白表达均低于ALLO+PBS+LIFU组和ALLO+NBs组(均P<0.05),ALLO+NBs+LIFU组急性排斥反应分级均明显低于ALLO+PBS+LIFU组及ALLO+NBs组(均P<0.05)。结论超声引导下携galectin-7-siRNA靶向微泡爆破治疗能降低大鼠移植心脏galectin-7表达,抑制急性排斥反应发生。     

Ultrasound Improves the Uptake and Distribution of Liposomal Doxorubicin in Prostate Cancer Xenografts

Combining liposomally encapsulated cytotoxic drugs with ultrasound exposure has improved the therapeutic response to cancer in animal models; however, little is known about the underlying mechanisms. This study focused on investigating the effect of ultrasound exposures (1 MHz and 300 kHz) on the delivery and distribution of liposomal doxorubicin in mice with prostate cancer xenografts. The mice were exposed to ultrasound 24 h after liposome administration to study the effect on release of doxorubicin and its penetration through the extracellular matrix. Optical imaging methods were used to examine the effects at both microscopic subcellular and macroscopic tissue levels. Confocal laser scanning microscopy revealed that ultrasound-exposed tumors had increased levels of released doxorubicin compared with unexposed control tumors and that the distribution of liposomes and doxorubicin through the tumor tissue was improved. Whole-animal optical imaging revealed that liposomes were taken up by both abdominal organs and tumors.     

Theoretical and experimental characterisation of magnetic microbubbles

In addition to improving image contrast, microbubbles have shown great potential in molecular imaging and drug/gene delivery. Previous work by the authors showed that considerable improvements in gene transfection efficiency were obtained using microbubbles loaded with magnetic nanoparticles under simultaneous exposure to ultrasound and magnetic fields. The aim of this study was to characterise the effect of nanoparticles on the dynamic and acoustic response of the microbubbles. High-speed video microscopy indicated that the amplitude of oscillation was very similar for magnetic and nonmagnetic microbubbles of the same size for the same ultrasound exposure (0.5 MHz, 100 kPa, 12-cycle pulse) and that this was minimally affected by an imposed magnetic field. The linear scattering to attenuation ratio (STAR) was also similar for suspensions of both bubble types although the nonlinear STAR was ~50% lower for the magnetic microbubbles. Both the video and acoustic data were supported by the results from theoretical modelling.     

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.