Name: production of membrane-filtered phase-shift decafluorobutane nanodroplets from preformed microbubbles
Uploaded: 2021-03-23T14:00:00
Description: Watch this Scientific Journal Video about Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles at JoVE.com

我们要感谢Ken Hoyt博士实验室的Dominique James为可蒸发的相移纳米滴提供TRSP分析。

1. 制作脂质薄膜
<ol><li>使用90%DSPC和10%DSPE-PEG2K制备用于微气泡生成的脂质薄膜，方法是使用以下方向以正确的比例混合脂质：<ol>
<li>在氯仿中制造DSPC和DSPE-PEG2K的储备脂质。在单独的小瓶中称量50mg每种脂质粉末。使用1 mL玻璃注射器向每个小瓶中加入1 mL氯仿。</li>
		<li>使用玻璃注射器将287μLDSPC储备液和113μLDSPE-PEG2K储备液（均为50mg / mL）加入20mL闪烁小瓶中。</li>
		<li>用氮气干燥混合脂质以除去氯?...

<ol>
	<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 Science Engineering and Technology. 1 (1-2), 3-17 (2009).</li><li>Sheeran, P. S., Dayton, 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=Phase-change+contrast+agents+for+imaging+and+therapy.">Phase-change contrast agents for imaging and therapy.</a> Current Pharmaceutical Design. 18 (15), 2152-2165 (2012).</li><li>Mountford, P. A., Smith, W. S., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorocarbon+nanodrops+as+acoustic+temperature+probes.">Fluorocarbon nanodrops as acoustic temperature probes.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 31 (39), 10656-10663 (2015).</li><li>Mountford, P. A., Thomas, A. N., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+activation+of+superheated+lipid-coated+perfluorocarbon+drops.">Thermal activation of superheated lipid-coated perfluorocarbon drops.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 31 (16), 4627-4634 (2015).</li><li>Sheeran, P. S., Luois, S., Dayton, P. A., Matsunaga, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+acoustic+studies+of+a+new+phase-shift+agent+for+diagnostic+and+therapeutic+ultrasound.">Formulation and acoustic studies of a new phase-shift agent for diagnostic and therapeutic ultrasound.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 27 (17), 10412-10420 (2011).</li><li>Sheeran, P. S., Dayton, 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=Improving+the+performance+of+phase-change+perfluorocarbon+droplets+for+medical+ultrasonography:+current+progress,+challenges,+and+prospects.">Improving the performance of phase-change perfluorocarbon droplets for medical ultrasonography: current progress, challenges, and prospects.</a> Scientifica. 2014, 579684 (2014).</li><li>Sheeran, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+generating+submicrometer+phase-shift+perfluorocarbon+droplets+for+applications+in+medical+ultrasonography.">Methods of generating submicrometer phase-shift perfluorocarbon droplets for applications in medical ultrasonography.</a> IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control. 64 (1), 252-263 (2017).</li><li>Sheeran, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decafluorobutane+as+a+phase-change+contrast+agent+for+low-energy+extravascular+ultrasonic+imaging.">Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging.</a> Ultrasound in Medicine &amp; Biology. 37 (9), 1518-1530 (2011).</li><li>de Gracia Lux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+for+the+formation+of+monodisperse+superheated+perfluorocarbon+nanodroplets+as+activatable+ultrasound+contrast+agents.">Novel method for the formation of monodisperse superheated perfluorocarbon nanodroplets as activatable ultrasound contrast agents.</a> RSC Advances. 7 (77), 48561-48568 (2017).</li><li>Mountford, P. A., Sirsi, S. R., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condensation+phase+diagrams+for+lipid-coated+perfluorobutane+microbubbles.">Condensation phase diagrams for lipid-coated perfluorobutane microbubbles.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 30 (21), 6209-6218 (2014).</li><li>Feshitan, J. A., Chen, C. C., Kwan, J. J., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+size+isolation+by+differential+centrifugation.">Microbubble size isolation by differential centrifugation.</a> Journal of Colloid and Interface Science. 329 (2), 316-324 (2009).</li><li>Wu, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ultrasound-facilitated+brain+drug+delivery+using+optimized+nanodroplets:+vaporization+efficiency+dictates+large+molecular+delivery.">Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets: vaporization efficiency dictates large molecular delivery.</a> Physics in Medicine and Biology. 63 (3), 035002 (2018).</li><li>Li, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+Nucleation+of+stable+perfluorocarbon+emulsions+for+ultrasound+contrast+agents.">Spontaneous Nucleation of stable perfluorocarbon emulsions for ultrasound contrast agents.</a> Nano Letters. 19 (1), 173-181 (2019).</li><li>Sheeran, P. S., Luois, S. H., Mullin, L. B., Matsunaga, T. O., Dayton, 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=Design+of+ultrasonically-activatable+nanoparticles+using+low+boiling+point+perfluorocarbons.">Design of ultrasonically-activatable nanoparticles using low boiling point perfluorocarbons.</a> Biomaterials. 33 (11), 3262-3269 (2012).</li><li>Kawabata, K., Sugita, N., Yoshikawa, H., Azuma, T., Umemura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+with+multiple+perfluorocarbons+for+controllable+ultrasonically+induced+phase+shifting.">Nanoparticles with multiple perfluorocarbons for controllable ultrasonically induced phase shifting.</a> Japanese Journal of Applied Physics. 44 (6), 4548-4552 (2005).</li><li>Shakya, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaporizable+endoskeletal+droplets+via+tunable+interfacial+melting+transitions.">Vaporizable endoskeletal droplets via tunable interfacial melting transitions.</a> Science Advances. 6 (14), 7188 (2020).</li><li>Kopechek, J. A., Zhang, P., Burgess, M. T., Porter, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+phase-shift+nanoemulsions+with+narrow+size+distributions+for+acoustic+droplet+vaporization+and+bubble-enhanced+ultrasound-mediated+ablation.">Synthesis of phase-shift nanoemulsions with narrow size distributions for acoustic droplet vaporization and bubble-enhanced ultrasound-mediated ablation.</a> Journal of Visualized Experiments: JoVE. (67), e4308 (2012).</li></ol>

有一套全面的文献，讨论了微气泡和相移液滴在体内成像和治疗中的配方、物理原理和潜在应用。该讨论明确涉及产生脂质微气泡，并使用低沸点DFB气体和高压挤出将其转化为亚微米相移液滴。这里概述的方法旨在通过将先前的微气泡缩合方法与液滴挤出相结合，提供一种相对简单的生产大量脂质微气泡和DFB相移液滴的方法。这种方法具有产生高浓度气泡的优点，用于形成基于过滤器选择的具有?...

超声造影剂（UCA）在成像和治疗应用中迅速普及。微气泡是最初的UCAs，目前是临床诊断应用中使用的主流药物。微气泡是充满气体的球体，通常直径为1-10μm，周围是脂质、蛋白质或聚合物壳1。然而，它们的大小和体内稳定性会限制它们在许多应用中的功能。相移纳米滴含有过热的液芯，由于其尺寸更小，循环寿命更高，因此可以克服其中一些限制2。当暴露于热或声能时，过热的液芯蒸发形成气体微气泡2，3，4，5。由于汽化阈值与液滴大小5，6直接相关，因此配制大小均匀的液滴悬浮液对于实现一致的活化阈值是非常理想的。产生均匀液滴尺寸的配方方法通常复杂且成本高昂，而更具成本效益的方法则会产生多分散解决方案7。另一个限制是能够产生具有低沸点全氟化碳（PFC）气体的稳定相移液滴，这对于体内高效活化至关重要8。在这份手稿中，描述了一种方案，用于产生稳定的过滤的低沸点可汽化相移液滴，用于体内成像和治疗应用。
生产单分散亚微米相移液滴的方法有很多7。控制尺寸的最可靠方法之一是使用微流体装置。这些设备可能成本高昂，液滴产生速度慢（约104-106滴/秒）7，并且需要广泛的培训。微流体装置通常还需要高沸点气体，以避免系统自发汽化和堵塞7。然而，de Gracia Lux等人最近的一项研究表明，如何使用低沸点十氟丁烷（DFB）或八氟丙烷（OFP）冷却微流体来产生高浓度的亚微米相移（1010-1012 / mL）。
通常，低沸点气体（如DFB或OFP）使用预制气泡更容易处理。前体脂质稳定的气泡可以通过使用低温和高压冷凝气体来产生可蒸发的液滴5，10。使用该方法产生的液滴浓度取决于前体微气泡浓度和气泡转化为液滴的效率。据报道，从接近1010 MB / mL11的尖端超声处理 >中报告了浓缩微气泡，而另一项研究报告了来自冷凝的OFP和DFP气泡的约1-3 x1011 液滴浓度12。当单分散液滴不是问题时，冷凝方法是使用低沸点PFC产生脂质稳定相移液滴的最直接，成本最低的方法。在冷凝前产生均匀尺寸气泡的方法可以帮助创建更多的单分散液滴群。然而，产生单分散的前体气泡也很困难，需要更昂贵的方法，如微流体或重复的差异离心技术11。最近发表了一种使用脂质体中液滴的自发成核来生产DFB和OFB纳米滴的替代方法13。这种方法利用"茴香烈酒"效应，是一种产生低沸点PFC液滴的简单方法，无需冷凝气泡。PFC液滴的大小分布可以通过精细滴定和混合用于启动液滴成核的PFC，脂质和乙醇组分来控制。同样值得注意的是，全氟化合物的混合可用于控制纳米滴的稳定性和活化阈值14，15。Shakya等人最近的工作证明了如何通过乳化碳氢化合物内骨骼中的高沸点PFC来调节纳米液滴活化，以促进液滴核心内的异质成核16，这是一种可以与其他形式的液滴尺寸过滤一起考虑的方法。
一旦形成，相移液滴可以在形成后挤出，以产生更多的单分散种群。事实上，Kopechek等人之前已经发表了与这里描述的方法类似的协议，使用 高沸点十二氟戊烷（DDFP）作为液滴核心。希望使用具有高沸点全氟化碳（在室温下稳定）的相移液滴的读者应参考上述文章。产生和挤出具有低沸点气体的液滴（如DFB和OFP）更为复杂，最好通过冷凝预制气泡来处理。
在该协议中，描述了使用探针尖端超声处理用DFB气芯生成预制脂质微气泡的常用方法。接下来，使用商用挤出机将预制的微气泡冷凝成亚微滴（图1）。然后，产生的液滴可以通过热和超声波激活。与传统的缩合方法相比，该方法可以产生更大体积的纳米滴溶液，具有更窄的尺寸分布，而无需昂贵的微流体装置。生产具有窄尺寸分布的纳米滴溶液可能会产生更均匀的汽化阈值。这将最大限度地发挥它们在成像、消融、药物递送和栓塞等众多应用中的潜力1，3，4，6。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62203/62203fig01.jpg"> 图1：用于将预制微气泡冷凝成相移纳米滴的高压挤出装置示意图。 微气泡溶液被添加到挤出机腔室中并包含在挤出机腔室中，并且从氮气罐中通过腔室进气阀施加250 psi。氮气将推动微气泡溶液通过腔室底部的过滤器，将样品冷凝成纳米滴。溶液最终通过样品出口管被挤出机推出并收集。 <a href="https://www.jove.com/files/ftp_upload/62203/62203fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>Collecting and centrifuging droplets</td>
		</tr>
		<tr>
			<td>200 nm polycarbonate filter</td>
			<td>Whatman</td>
			<td>110606</td>
			<td>Extruder filters</td>
		</tr>
		<tr>
			<td>2-methylbutane</td>
			<td>Fisher Chemical</td>
			<td>03551-4</td>
			<td>Rapid precooling of microbubble solution prior to extrusion</td>
		</tr>
		<tr>
			<td>3-prong clamps X2</td>
			<td>Fisher</td>
			<td>02-217-002</td>
			<td>Holding scintilation vials in place for probe tip sonication</td>
		</tr>
		<tr>
			<td>400W Analog Probe Tip Sonicator with Horn</td>
			<td>Branson</td>
			<td>101-063-198R</td>
			<td>Used to generate lipid microbubbles from lipid solution</td>
		</tr>
		<tr>
			<td>Bath Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337402</td>
			<td>Used to help breakdown liposomes into unilamellar vesicles</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Bioreagents</td>
			<td>C298-4</td>
			<td>Used to make lipid film for microbubble preperation</td>
		</tr>
		<tr>
			<td>Decafluorobutane (Perfluorobutane) Gas</td>
			<td>FluoroMed L.P.</td>
			<td>1 kg</td>
			<td>generating microbubbles via probe tip sonication</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>-</td>
			<td>-</td>
			<td>Rapid precooling of microbubble solution prior to extrusion</td>
		</tr>
		<tr>
			<td>DSPC Lipid Powder</td>
			<td>NOF America</td>
			<td>COATSOME MC-8080</td>
			<td>Component of lipid film</td>
		</tr>
		<tr>
			<td>DSPE-PEG-2K Lipid Powder</td>
			<td>NOF America</td>
			<td>SUNBRIGHT DSPE-020CN</td>
			<td>Component of lipid film</td>
		</tr>
		<tr>
			<td>General Thermometer</td>
			<td>-</td>
			<td>-</td>
			<td>Used to measure ice bath temperature and 2-methylbutane temperature ( needs to accommodate -20C temperatures)</td>
		</tr>
		<tr>
			<td>Glass Syringes</td>
			<td>Hamilton</td>
			<td>81139</td>
			<td>Used to mix lipids in chloroform</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Bioreagents</td>
			<td>BP229-1</td>
			<td>Reduces freezing temperature of PBS solution</td>
		</tr>
		<tr>
			<td>Heating Block</td>
			<td>VWR Scientific Products</td>
			<td></td>
			<td>Heating lipid films and vaporizing droplets</td>
		</tr>
		<tr>
			<td>Lipex 10 mL Extruder</td>
			<td>Evonik</td>
			<td></td>
			<td>Commercial high-pressure extrusion system</td>
		</tr>
		<tr>
			<td>Mini Vortex Mixer</td>
			<td>Fisher brand</td>
			<td>14-955-151</td>
			<td>Used to remove excess chloroform from lipid films</td>
		</tr>
		<tr>
			<td>Nitrogen Tank</td>
			<td>-</td>
			<td>-</td>
			<td>Used to operate extruder</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Hydrate lipid films and washing droplets</td>
		</tr>
		<tr>
			<td>Polyester Drain Disk</td>
			<td>Whatman</td>
			<td>230600</td>
			<td>Provides support for polycarbonate filter</td>
		</tr>
		<tr>
			<td>Polypropylene Caps</td>
			<td>Fisher Scientific</td>
			<td>298417</td>
			<td>Used for solution storage</td>
		</tr>
		<tr>
			<td>Propylene Glycol</td>
			<td>Fisher Chemical</td>
			<td>P355-1</td>
			<td>Reduces freezing temperature of PBS solution</td>
		</tr>
		<tr>
			<td>Scintiliation Vials</td>
			<td>DWK Life Sciences Wheaton</td>
			<td>986532</td>
			<td>Used for lipid films and microbubble generation</td>
		</tr>
		<tr>
			<td>Small hammer</td>
			<td>-</td>
			<td>-</td>
			<td>Used to break apart dry ice for cooling methylbutane</td>
		</tr>
		<tr>
			<td>Sonicator Microtip Attachment</td>
			<td>Branson</td>
			<td>101148070</td>
			<td>Used to generate microbubbles from lipid solution</td>
		</tr>
		<tr>
			<td>Steel Container</td>
			<td>Medegen</td>
			<td>79310</td>
			<td>Rapid precooling of microbubble solution prior to extrusion ( any container rated to -20C will work)</td>
		</tr>
		<tr>
			<td>Vacuume Dessicator</td>
			<td>Bel-Art SP Scienceware</td>
			<td>08-648-100</td>
			<td>Removes excess chloroform from lipid films</td>
		</tr>
		<tr>
			<td>2mL Centrifuge Tube</td>
			<td>Fisher</td>
			<td>02682004</td>
			<td>Used for concentrating nanodroplets </td>
		</tr>
	</tbody>
</table>

production of membrane-filtered phase-shift decafluorobutane nanodroplets from preformed microbubbles

有许多方法可用于生产用于成像和治疗的可蒸发相移液滴。每种方法都使用不同的技术，并且在价格，材料和目的方面有所不同。许多这些制造方法导致具有不均匀激活阈值的多分散群体。此外，控制液滴大小通常需要稳定的全氟碳液体，其高活化阈值在体内是不切实际的。使用低沸点气体产生均匀的液滴尺寸将有利于体内成像和治疗实验。本文介绍了一种简单而经济的方法，用于形成具有低沸点十氟丁烷（DFB）的尺寸过滤脂质稳定的相移纳米滴。描述了产生脂质微气泡的常用方法，以及一种在单个步骤中用高压挤出冷凝它们的新方法。该方法旨在节省时间，最大限度地提高效率，并使用许多生物实验室中的常见实验室设备为各种应用生成更大体积的微气泡和纳米滴解决方案。

使用动态光散射（DLS）和可调阻脉冲检测（TRSP）分析，包括尺寸分布的代表性结果。图5显示了有和不带挤出的冷凝气泡溶液的尺寸分布。在没有挤出的情况下，实验方案在步骤5.3结束。冷却的气泡通过在冷时将样品排放到大气压来冷凝。仅浓缩的样品具有更宽的分布，集中在400nm附近。挤出的样品具有较窄的分布，集中在200nm处。两个样本都包括脂质体和液滴，使用DLS无法?...

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Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

该协议描述了一种使用探针尖端超声处理产生大量脂质封装的十氟丁烷微气泡的方法，然后使用高压挤出和机械过滤将它们冷凝成相移纳米滴。

从预成型微气泡生产膜过滤相移十氟丁烷纳米滴

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

该协议是减少低沸点可汽化液滴多分散性的简单方法，用于生物医学应用。该技术以可控、可扩展且相对经济高效的方式冷凝预制的气体微气泡，并按尺寸过滤产生的液滴。该方法可应用于具有不同壳体和气体材料的各种脂质微泡，使用不同的过滤器来控制最终液滴的大小。演示该程序的将是Darrah Merillat，他是来自Dr.Sirsi实验室的本科生研究员。打开声波器的电源开关，并使用微针附件将振幅设置为允许的最大值，并确保超声处理时间设置为10秒。将温水合脂质溶液放入隔音罩中，微针位于表面下方。将适当长度的管子连接到小瓶的颈部，以引导气体从DFB罐出口进入外壳中保存的温脂溶液中。缓慢打开罐阀，直到可以看到气体流过脂质溶液，在液体表面引起轻微的涟漪。如果气体流量过高，溶液将在微气泡形成过程中溢出。启动超声仪，连续运行10秒钟以产生微气泡。超声处理结束后，立即关闭DFB罐阀。快速盖上微气泡溶液，并将小瓶浸没在冰浴中，将样品冷却到55摄氏度以下。使用200纳米陶瓷过滤器根据用户手册组装高压挤出机，并将其放置在防水容器的中心，这样样品出口管就不会压在侧面或卷曲。使用制造商提供的适配器将挤出机连接到氮气罐。将出口管的末端置于闪烁小瓶中以收集挤出的样品，用胶带将管固定在容器中以保持在小瓶内。打开和关闭释放阀，以确保挤出机内没有压力。取下腔室盖。并将五毫升PBS加入挤出机室。盖上盖子，确保盖子卡回原位。打开氮气罐，使压力表读数为250 PSI，确保压力控制阀处于关闭位置。关闭气罐，打开挤出机腔进气阀，使PBS溶液被推过系统，并将样品出口管排出进入闪烁小瓶。当只有气体从管道中流出时，打开释放阀，让压力降至零PSI。然后取出闪烁小瓶。打开和关闭释放阀，以确保挤出机内没有压力，并在出口管的末端放置一个新的闪烁小瓶。在钢制容器中装满2-甲基丁烷，并加入干冰，将温度降至零下18摄氏度。将微气泡溶液插入冷却的2-甲基丁烷中，浸没样品两分钟。在两分钟内移动闪烁小瓶，轻轻混合气泡。根据需要添加干冰，以保持温度在零下15摄氏度至零下18摄氏度之间。两分钟后，从冷藏的2-甲基丁烷中除去微气泡。轻轻旋转小瓶以混合微气泡，并将气泡转移到冷却的10毫升注射器中。取下挤出机腔盖，并通过缓慢推动注射器上的柱塞将微泡溶液加入腔室中。更换挤出机盖，确保其卡入到位。验证挤出机的压力控制阀和释放阀是否处于关闭位置。打开氮气罐，直到压力表读数为250 PSI。关闭油箱，将压力控制阀转动至打开位置。当溶液在出口管处填充闪烁小瓶并且只有气体离开管时，慢慢打开压力释放阀，并使压力降至零PSI。将10毫升挤出的液滴溶液转移到15毫升的离心管中。将挤出的样品在4摄氏度下以1， 500 g离心10分钟。丢弃上清液和出现在溶液顶部的自发汽化液滴。将包含DFP纳米滴的沉淀重新悬浮在10毫升PBS中，其中含有20%甘油和20%丙二醇。有和不带挤出的冷凝气泡溶液的尺寸分布表明，仅浓缩的样品具有更宽的分布中心，中心在400纳米附近，而挤出样品的分布范围较窄，集中在200纳米。用于分析相移液滴的可调谐电阻脉冲传感分析，因为它们已被离心洗涤以除去多余的脂质体，表明液滴尺寸接近200纳米。加热时纳米滴蒸发的显微镜数据显示，加热前视野中一些自发蒸发的微气泡明显，加热后观察到的气体微气泡数量较多。这里显示了直接插入挤出机的纳米滴的显微镜图像，无需预冷，在零摄氏度和零下18摄氏度下冷凝。汽化前后凝聚的八氟丙烷液滴的代表性图像也显示加热后气体微气泡数量更多，类似于DFB液滴。在此过程中要记住的最重要的事情是，液滴产量高度依赖于冷凝过程中的温度和压力，微小的变化会影响结果。在产生可蒸发的液滴后，它们可用于优化体内成像和使用超声波的药物输送，以及其他体内和离体应用。在开发该技术后，已经通过对该协议的轻微修改来探索纳米滴尺寸和气体核心含量对体内汽化阈值的影响。

Research

JoVE Journal

Bioengineering

Production and Targeting of Monovalent Quantum Dots

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

科研

生物工程

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

化学品生产<em&gt;肺炎克雷伯菌</em使用竹水合物作为原料

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

含脂脂的微流体生产-温度敏感脂质体

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

使用高速摄像机和图像分析对快速移动微泡区域进行成像和定量

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

An Efficient Method for Adenovirus Production

腺病毒生产的有效方法

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

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

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 ...