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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

15.5K 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

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

发育生物学

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

医学

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

声学悬浮系统中非球形气泡振荡诱导微流

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

工程学

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy platforms. When combined with therapeutic focused ultrasound (FUS), they can safely and locally overcome physiological barriers (e.g., blood-brain barrier), deliver drugs to otherwise inaccessible cancers (e.g., glioblastoma and pancreatic cancer), and treat neurodegenerative diseases. The therapeutic arsenal of microbubble-FUS is advancing in new directions, including synergistic combination radiotherapy, multimodal imaging, and all-in-one drug loading and delivery from microbubble shells.
Labeling microbubbles with radiotracers is key to establishing these expanded theranostic capabilities. However, existing microbubble radiolabeling strategies rely on purification methodologies known to perturb microbubble physicochemical properties, use short-lived radioisotopes, and do not always yield stable chelation. Collectively, this creates ambiguity surrounding the accuracy of microbubble radioimaging and the efficiency of tumor radioisotope delivery.
This protocol describes a new one-pot, purification-free microbubble labeling methodology that preserves microbubble physicochemical properties while achieving &#62;95% radioisotope chelation efficiency. It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length, charge, and chelator/probe (porphyrin, DTPA, DiI) composition. It can be adaptively applied during ground-up microbubble fabrication and to pre-made microbubble formulations with modular customizability of fluorescence and multimodal fluorescence/radioactive properties. Accordingly, this flexible method enables the production of tailored, traceable (radio, fluorescent, or radio/fluorescent active) multimodal microbubbles that are useful for advancing mechanistic, imaging, and therapeutic microbubble-FUS applications.

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

使用荧光和放射性示踪剂制造和标记微气泡

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

癌症研究

Studying Cavitation Enhanced Therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

研究屈化强化疗法

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

实时活体内多光子显微镜可可视化聚焦超声和微气泡处理，以提高血脑屏障通透性

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

用于表征用于超声触发药物释放的荧光标记微气泡的多时间尺度显微镜方法

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...