格兰特支持包括NHLBI HL080532，NHLBI HL073449，NCRR RR16453的AHA国家批准的援助奖（RVS）。特别感谢延长距离的课程设计和咨询组（DCDC），dcdcgroup.org，协助视频制作和美国能源部的教育补助金号P336C050047创办的DCDC。

1。微气泡的股票准备<ol><li>在10毫升的PBS混合1克葡萄糖200毫克1,2 - 二棕榈酰- SN -甘油-3 - 磷脂和50毫克1,2 - 二棕榈酰- SN -甘油-3 - 磷脂。 </li><li>在沸水浴20-30分钟中加热的混合物，吸管搅拌，每隔5分钟。 </li><li>该解决方案可以存储在4℃至6个月。 </li></ol> 2。微泡的制备<ol><li>以250μL的准备微泡造影剂原液和孵化，在40 ° 15分钟。 </li><li>预热的微泡造影剂的解决方案，然后转移到1.5 mL微管含50μL的甘油。 </li><li>纯化的质粒DNA编码的表达式构造感兴趣的基因（Qiagen公司的内毒素MegaPrep试剂盒，QIAGEN，日耳曼，医学博士，在这个例子中纯化的最佳浓度4mg/ml）1-2毫克。 2.4）磷酸盐缓冲液加入到500μL的最终体积。内毒素Qiagen公司maxipreps使用，以及无菌PBS，以保证无菌。 </li><li>在微管中的空气是那么Octafluoropropane天然气取代。 </li><li>微管，然后动摇大力牙科混汞为20秒。 </li><li> subnatant含有残留DNA和缓冲区，没有约束的微泡，然后小心取出，微气泡层是用无菌PBS洗涤三次删除独立的DNA，并置于冰之间的每个洗涤周期。我们通常达到30-40％的约束力的效率。所有试剂都是无菌和护理，以尽量减少污染。 </li><li>质粒DNA结合微泡，然后放在冰长达两个小时，直到使用。 </li><li> subnatant从混合后，PBS洗涤的微管删除，可以被用来确定未结合DNA的浓度，同样的金额势必根据已知的初始浓度，在使用260nm波长，这种解决方案的光密度测量分光光度计。 </li></ol> 3。设备校准<ol><li>第一次使用之前，1 MHz的空蚀传感器需要进行校准，以确保适当的机械指数和脉冲重复。一个submergible 1兆赫，为13mm，重点不突出传感器连接到一个20 MHz函数/任意波形发生器，通过一个功率放大器。 </li><li>该传感器被放置在一个装满水的塑料容器，在一个水听器，已通过电荷放大器连接到500 MHz示波器的直接目的。 </li><li>波形，频率，振幅，突发周期，和功率放大都可以进行修改，以获得正确的的占空比和机械指数的最佳气蚀的微泡。对于这个特殊的实验中，我们已校准系统，机械指数在1 MHz〜1.3。 </li></ol> 4。微泡配送及UTMD <ol><li>微泡造影剂的交付和UTMD之前，C57BL / 6小鼠分别通过腹腔注射100mg/kg氯胺酮和为5mg/kg甲苯噻嗪麻醉。 </li><li>微气泡的交付是通过直接注射给药，静脉注射进入心脏的左心室或通过尾静脉插管。对于直接注入心脏，量高达100μL的质粒DNA加载微泡造影剂的解决方案是通过30号针插在超声波作用下的可视化前的第 4肋间进入心脏的左心室注入。 </li><li>从左边的室间注射微泡的解决方案丸是用视觉超音速队的38 MHz的高频超声换能器放置在固定位置上的鼠标在长轴使用VisualSonics胸部“VEVO 2100成像系统的可视化。所有的注射器和针头是无菌的，投保最无菌的环境可能对这些注射。 </li><li>紧随注射，微气泡的破坏进行〜5分钟，第二，小尺寸的低频率为1.0 MHz传感器直接持有超过所需的器官，针对本地区的破坏。在这个例子中，超声是管理的肝脏在脉冲重复频率为1.0 MHz，机械指数相当于约1.3-1.5，100毫秒脉冲重复每20个周期内。另外，脉冲可门控鼠标心电图一阵超声3张，每4-6个心动周期（在本实验中未显示）。我们取得更大的转染效率与协议，使毛细血管床之间的阵阵超声气泡笔芯。 </li></ol> 5。另类送货方法我们选择了突出的室间注射，由于程序复杂，但在许多情况下，如长期输液的微泡，尾静脉，jection是首选方法。对于微泡交付的尾部静脉方法，鼠标麻醉方式相同。注射器含有质粒DNA结合微泡，是连接到一个27号针头/尾静脉导管。尾静脉导管插入远端三分之一的向左或向右，鼠标的尾部沿侧脉。是摆在输液泵，自动管理一个统一的解决方案预置量超过预设的时间内内含有微气泡的注射器。我们通常在3ml/hour率注入200 -300μL。 动物用 处理所有动物均按照有关国家和/或当地的动物福利机构定义在具有良好的动物实践，和所有动物的工作是适当的委员会批准（夏威夷机构动物护理和使用委员会大学，批准文号07-100 -3）。使用适当的麻醉（氯胺酮/ zylazine）和止痛药（布比卡因和丁丙诺啡），尽管不要求。 6。代表性的成果： UTMD介导质粒DNA交付的有效性进行评估可以通过多种方法，取决于在构造，如编码的基因，但不限于在活体成像，B -半乳糖苷酶体外染色，和/或萤光素酶免疫组化。特别是，允许一个监控基因表达的存在和持续时间在一个发光的记者（荧光素酶）基因与质粒转染小鼠连续在体内生物发光成像。精诺真活体成像系统（IVIS）（卡尺生命科学，霍普金顿，马）是用于发光成像。图片通常采取UTMD介导转后的第一天所有小鼠和重复，每三到四天，直到发光基因的表达不再是系统（图1）通过视觉检测。要准备发光成像的小鼠，小鼠第一次收到的荧光素酶报告的IP注入探头的D -荧光素（卡尺生命科学版），然后麻醉〜3分钟后。 〜10分钟出发前的动物是摆在IVIS成像室和一个完整的人体图像扫描的D -荧光素底物的生物分布。在采集过程中，萤火虫荧光素酶/ D -荧光素的光化学反应发出的光子进行测量。图1也说明了类似IVIS肝脏发光成像以下UTMD，图2是一个epifluorescence（100X）转染肝的形象，使用抗荧光素酶抗体（Sigma - Aldrich公司）和AlexFluor - 568标记的二抗（Invitrogen公司） 。很清楚地看到，UTMD介导的肝转不仅影响了血管内皮细胞，但hepatocyes。 <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="图1" /> 图1（一）负控制鼠标：精诺真/ IVIS心脏成像UTMD治疗的小鼠。质粒+ PBS心脏指示UTMD，（b）治疗鼠标：质粒+通过心脏的微泡指示UTMD，和（C）治疗鼠标UTMD质粒+由肝脏微泡指示。 <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="图2" /> 图（一）2。肝脏免疫组织化学UTMD“（红色反荧光素酶）。质粒与没有UTMD，及（B）与UTMD质粒。共聚焦图像（100X）;细胞核DAPI染色蓝。

<ol>
	<li>Bekeredjian, R., Chen, S., Frenkel, P. A., Grayburn, P. A., Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+can+repeatedly+direct+highly+specific+plasmid+expression+to+the+heart.">Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart.</a> Circulation. 108, 1022-1026 (2003).</li><li>Bekeredjian, R., Katus, H. A., Kuecherer, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+ultrasound+targeted+microbubble+destruction:+a+review+of+non-cardiac+applications.">Therapeutic use of ultrasound targeted microbubble destruction: a review of non-cardiac applications.</a> Ultraschall Med. 27, 134-140 (2006).</li><li>Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+pancreatic+islets+in+vivo+by+ultrasound-targeted+gene+therapy.">Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.</a> Gene Ther. 17, 1411-1420 (2010).</li><li>Miao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+enhances+gene+delivery+of+human+factor+IX+plasmid.">Ultrasound enhances gene delivery of human factor IX plasmid.</a> Hum Gene Ther. 16, 893-905 (2005).</li><li>Shimoda, M., Chen, S., Noguchi, H., Matsumoto, S., Grayburn, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+non-viral+gene+delivery+of+human+vascular+endothelial+growth+factor+improves+revascularisation+and+restoration+of+euglycaemia+after+human+islet+transplantation+into+mouse+liver.">In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.</a> Diabetologia. 53, 1669-1679 (2010).</li><li>Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+destruction+of+albumin+microbubbles+directs+gene+delivery+to+the+myocardium.">Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.</a> Circulation. 101, 2554-2556 (2000).</li><li>Sirsi, S., Borden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+Compositions,+Properties+and+Biomedical+Applications.">Microbubble Compositions, Properties and Biomedical Applications.</a> Bubble Sci Eng Technol. 1, 3-17 (2009).</li><li>Li, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+enhances+AAV-mediated+gene+transfection+in+human+RPE+cells+in+vitro+and+rat+retina+in+vivo.">Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo.</a> Gene Ther. 16, 1146-1153 (2009).</li><li>Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Newman, C. M., Bettinger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+ultrasound+for+gene+transfer.">Gene therapy progress and prospects: ultrasound for gene transfer.</a> Gene Ther. 14, 465-475 (2007).</li><li>Vancraeynest, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+injury+induced+by+ultrasound-targeted+microbubble+destruction:+evidence+for+the+contribution+of+myocardial+ischemia.">Myocardial injury induced by ultrasound-targeted microbubble destruction: evidence for the contribution of myocardial ischemia.</a> Ultrasound Med Biol. 35, 672-679 (2009).</li></ol>

UTMD代表一个基因传递的新方法。作为一个平台技术，它可以与许多潜在的基因治疗策略相结合，提供无数时需要高度的组织特异性的生物活性分子。生物技术的主要限制是转染效率低。另一个重要的考虑是方便的靶器官超声，可显着减少干预骨或空气。该技术需要优化技术的基础上靶组织11以及声学的一个基本的了解，以限制组织损伤 11，提高工作效率。但是，它提供了一种廉?...

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> 1,2 - 二棕榈酰- SN -甘油-3 - 磷脂</td><td> Sigma - Aldrich公司</td><td> P - 5911 </td><td>微泡造影剂的脂质外壳组件</td></tr><tr><td> 1,2 - 二棕榈酰- SN -甘油-3 - 磷脂</td><td> Sigma - Aldrich公司</td><td> P - 3275 </td><td>微泡造影剂的脂质外壳组件</td></tr><tr><td>葡萄糖</td><td> Sigma - Aldrich公司</td><td> G5400 </td><td>认为稳定微泡</td></tr><tr><td>磷酸盐缓冲液</td><td> Sigma - Aldrich公司</td><td> P5368 </td><td></td></tr><tr><td>甘油</td><td> Sigma - Aldrich公司</td><td> G5516 </td><td>相信，以防止凝聚微泡</td></tr><tr><td> Octafluoropropane气体</td><td>公司Airgas </td><td> N / A </td><td>在临床应用中所用的惰性气体</td></tr><tr><td> VialMix牙科混汞</td><td>施贵宝</td><td> N / A </td><td></td></tr><tr><td> 1 MHz时，为13mm，重点不突出传感器</td><td>奥林巴斯</td><td> A303S - SU </td><td></td></tr><tr><td> 20 MHz函数/任意波形发生器</td><td>安捷伦</td><td> 33220A </td><td></td></tr><tr><td>功率放大器</td><td>克罗恩-海特有限公司</td><td>型号7500 </td><td></td></tr><tr><td>水听器</td><td> Brüel和克亚尔的</td><td>类型1803 </td><td></td></tr><tr><td>电荷放大器</td><td> Brüel和克亚尔的</td><td>类型2634 </td><td></td></tr><tr><td> 500 MHz示波器</td><td>力科</td><td> 9354L </td><td></td></tr><tr><td> 2100 VisualSonics“VEVO 34兆赫传感器的成像系统</td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G一英寸尾静脉导管</td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td>精灵加输液泵</td><td>肯特科学</td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

UTMD，生物活性分子，如带负电荷的质粒DNA编码感兴趣的基因的载体，添加阳离子脂质微泡造影剂7-9炮弹。在小鼠中，这些载体携带的微泡可静脉给药，或直接向心脏的左心室。在较大的动物，他们也可以通过冠状动脉导管注入。随后的交付，从流通到靶器官，发生在微泡的共振频率的声空化。这很可能是产生机械能在短暂的或有针对性的地区的10个微血管内皮细胞之间的孔隙形成的微气泡的破坏结果。由于这种sonoporation效应的结果，和跨内皮细胞的转染效率的提高，和转基因的编码向量存放到周围组织。质粒DNA留在流通迅速降解血液中的核酸交货的可能性，从而进一步降低非超声组织和高度特异性的靶器官转。

Watch this Scientific Journal Video about Introduction to the Ultrasound Targeted Microbubble Destruction Technique at JoVE.com

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

超声针对性的微泡销毁（UTMD）可用于直接站点特定的生物活性分子的交付，包括基因治疗的靶器官，如心脏和肝脏超声，<sup&gt; 1-6</sup&gt;。

有针对性的超声微泡销毁技术简介

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

15.4K 次观看.  University of Hawaii. 超声针对性的微泡销毁（UTMD）可用于直接站点特定的生物活性分子的交付，包括基因治疗的靶器官，如心脏和肝脏超声， 1-6。

视频: Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

超声造影有针对性的治疗胶质瘤的小鼠通过药物轴承纳米粒子交付和微血管消融

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

分析有针对性的病毒蛋白的纳米粒子递送HER2 +肿瘤

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

制造浓缩，脂质为基础的氧气微泡的乳液由高剪切均质化和串行集中

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

基于 CMUT 技术的新型超声探头在脑成像中的应用研究

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...