Name: microfluidic production of lysolipid-containing temperature-sensitive liposomes
Uploaded: 2020-03-03T13:00:00
Description: Watch this Scientific Journal Video about Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes at JoVE.com

我们感谢英国前列腺癌（CDF-12-002奖学金）和工程和物理科学研究委员会（EPSRC）（EP/M008657/1）的资助。

1. 设备设置
<ol><li>组装注射器泵和 SHM，如下所示。<ol>
<li>使用泵到泵网络电缆（图1，黄色）将辅助注射器泵的"到计算机"端口连接到主注射器泵的"到网络"端口（泵01，用于乙醇脂质溶液）。</li>
		<li>使用 PC 泵网络电缆将主泵的"到计算机"端口连接到计算机的"RS232 串行"端口（图1，蓝色）。</li>
		<li>使用螺母和套圈将油管连?...

<ol>
	<li>Needham, D., Park, J., Wright, A. M., Tong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+characterization+of+the+low+temperature+sensitive+liposome+(LTSL):+effects+of+the+lipid+composition+(lysolipid+and+DSPE-PEG2000)+on+the+thermal+transition+and+release+of+doxorubicin.">Materials characterization of the low temperature sensitive liposome (LTSL): effects of the lipid composition (lysolipid and DSPE-PEG2000) on the thermal transition and release of doxorubicin.</a> Faraday Discussions. 161, 515-534 (2013).</li><li>Ickenstein, L. M., Arfvidsson, M. C., Needham, D., Mayer, L. D., Edwards, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disc+formation+in+cholesterol-free+liposomes+during+phase+transition.">Disc formation in cholesterol-free liposomes during phase transition.</a> Biochimica et Biophysica Acta - Biomembranes. 1614 (2), 135-138 (2003).</li><li>Valencia, P. M., Farokhzad, O. C., Karnik, R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+technologies+for+accelerating+the+clinical+translation+of+nanoparticles.">Microfluidic technologies for accelerating the clinical translation of nanoparticles.</a> Nature Nanotechnology. 7 (10), 623-629 (2012).</li><li>Chen, D., 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=Rapid+discovery+of+potent+siRNA-containing+lipid+nanoparticles+enabled+by+controlled+microfluidic+formulation.">Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation.</a> Journal of the American Chemical Society. 134 (16), 6948-6951 (2012).</li><li>Forbes, N., 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=Rapid+and+scale-independent+microfluidic+manufacture+of+liposomes+entrapping+protein+incorporating+in-line+purification+and+at-line+size+monitoring.">Rapid and scale-independent microfluidic manufacture of liposomes entrapping protein incorporating in-line purification and at-line size monitoring.</a> International Journal of Pharmaceutics. 556, 68-81 (2019).</li><li>Dittrich, P. S., Manz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-a-chip:+microfluidics+in+drug+discovery.">Lab-on-a-chip: microfluidics in drug discovery.</a> Nature Reviews Drug Discovery. 5 (3), 210-218 (2006).</li><li>Capretto, L., Carugo, D., Mazzitelli, S., Nastruzzi, C., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+and+lab-on-a-chip+preparation+routes+for+organic+nanoparticles+and+vesicular+systems+for+nanomedicine+applications.">Microfluidic and lab-on-a-chip preparation routes for organic nanoparticles and vesicular systems for nanomedicine applications.</a> Advanced Drug Delivery Reviews. 65 (11-12), 1496-1532 (2013).</li><li>Cheung, C. C. L., Al-Jamal, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterically+stabilized+liposomes+production+using+staggered+herringbone+micromixer:+Effect+of+lipid+composition+and+PEG-lipid+content.">Sterically stabilized liposomes production using staggered herringbone micromixer: Effect of lipid composition and PEG-lipid content.</a> International Journal of Pharmaceutics. 566, 687-696 (2019).</li><li>Suh, Y. K., Kang, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+Mixing+in+Microfluidics.">Review on Mixing in Microfluidics.</a> Micromachines. 1 (3), 82-111 (2010).</li><li>Jahn, A., Vreeland, W. N., Gaitan, M., Locascio, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Vesicle+Self-Assembly+in+Microfluidic+Channels+with+Hydrodynamic+Focusing.">Controlled Vesicle Self-Assembly in Microfluidic Channels with Hydrodynamic Focusing.</a> Journal of the American Chemical Society. 126 (9), 2674-2675 (2004).</li><li>Stroock, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaotic+Mixer+for+Microchannels.">Chaotic Mixer for Microchannels.</a> Science. 295 (5555), 647-651 (2002).</li><li>Belliveau, N. M., 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=Microfluidic+Synthesis+of+Highly+Potent+Limit-size+Lipid+Nanoparticles+for+In+Vivo+Delivery+of+siRNA.">Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA.</a> Molecular Therapy - Nucleic Acids. 1 (8), 37 (2012).</li><li>Patra, M., 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=Under+the+influence+of+alcohol:+The+effect+of+ethanol+and+methanol+on+lipid+bilayers.">Under the influence of alcohol: The effect of ethanol and methanol on lipid bilayers.</a> Biophysical Journal. 90 (4), 1121-1135 (2006).</li><li>Komatsu, H., Rowe, E. S., Rowe, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Cholesterol+on+the+Ethanol-Induced+Interdigitated+Gel+Phase+in+Phosphatidylcholine:+Use+of+Fluorophore+Pyrene-Labeled+Phosphatidylcholine.">Effect of Cholesterol on the Ethanol-Induced Interdigitated Gel Phase in Phosphatidylcholine: Use of Fluorophore Pyrene-Labeled Phosphatidylcholine.</a> Biochemistry. 30 (9), 2463-2470 (1991).</li><li>Lu, J., Hao, Y., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Cholesterol+on+the+in+Lysophosphatidylcholine+Formation+of+an+Interdigitated+Gel+Phase+and+Phosphatidylcholine+Binary.">Effect of Cholesterol on the in Lysophosphatidylcholine Formation of an Interdigitated Gel Phase and Phosphatidylcholine Binary.</a> Journal of Biochemistry. 129 (6), 891-898 (2001).</li><li>Vanegas, J. M., Contreras, M. F., Faller, R., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+unsaturated+lipid+and+ergosterol+in+ethanol+tolerance+of+model+yeast+biomembranes.">Role of unsaturated lipid and ergosterol in ethanol tolerance of model yeast biomembranes.</a> Biophysical Journal. 102 (3), 507-516 (2012).</li><li>Haran, G., Cohen, R., Bar, L. K., Barenholz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+ammonium+sulfate+gradients+in+liposomes+produce+efficient+and+stable+entrapment+of+amphipathic+weak+bases.">Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1151 (2), 201-215 (1993).</li><li>Sadeghi, N., 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=Influence+of+cholesterol+inclusion+on+the+doxorubicin+release+characteristics+of+lysolipid-based+thermosensitive+liposomes.">Influence of cholesterol inclusion on the doxorubicin release characteristics of lysolipid-based thermosensitive liposomes.</a> International Journal of Pharmaceutics. 548 (2), 778-782 (2018).</li><li>Lawaczeck, R., Kainosho, M., Chan, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+and+annealing+of+structural+defects+in+lipid+bilayer+vesicles.">The formation and annealing of structural defects in lipid bilayer vesicles.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 443 (3), 313-330 (1976).</li><li>Komatsu, H., Okada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol-induced+aggregation+and+fusion+of+small+phosphatidylcholine+liposome:+participation+of+interdigitated+membrane+formation+in+their+processes.">Ethanol-induced aggregation and fusion of small phosphatidylcholine liposome: participation of interdigitated membrane formation in their processes.</a> BBA - Biomembranes. 1235 (2), 270-280 (1995).</li><li>Marsh, D., Bartucci, R., Sportelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+membranes+with+grafted+polymers:+physicochemical+aspects.">Lipid membranes with grafted polymers: physicochemical aspects.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1615 (1-2), 33-59 (2003).</li><li>Hood, R. R., Vreeland, W. N., DeVoe, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+remote+loading+for+rapid+single-step+liposomal+drug+preparation.">Microfluidic remote loading for rapid single-step liposomal drug preparation.</a> Lab on a Chip. 14 (17), 3359-3367 (2014).</li><li>Dalwadi, G., Benson, H. A. E., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+diafiltration+and+tangential+flow+filtration+for+purification+of+nanoparticle+suspensions.">Comparison of diafiltration and tangential flow filtration for purification of nanoparticle suspensions.</a> Pharmaceutical Research. , (2005).</li><li>Roces, C., Kastner, E., Stone, P., Lowry, D., Perrie, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Quantification+and+Validation+of+Lipid+Concentrations+within+Liposomes.">Rapid Quantification and Validation of Lipid Concentrations within Liposomes.</a> Pharmaceutics. 8 (3), 29 (2016).</li><li>Kim, S. -. H., Kim, J. W., Kim, D. -. H., Han, S. -. H., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced-throughput+production+of+polymersomes+using+a+parallelized+capillary+microfluidic+device.">Enhanced-throughput production of polymersomes using a parallelized capillary microfluidic device.</a> Microfluidics and Nanofluidics. 14 (3-4), 509-514 (2013).</li></ol>

提出的协议描述了使用交错的刺骨微混频器（SHM）制备低温敏感脂质体（LTSLs）。LTSL10配方可在临床上达到的42°C高热温度下，在5分钟内释放多索鲁比辛。Indocyanine 绿色 （ICG） 也可以共同加载用于光热加热触发 DOX 的释放。该方法依赖于：（i） 在SHM11中乙醇和硫酸铵溶液的快速混沌混合所提供的均质溶剂环境下，将磷脂自行组装成脂质体;（二） 脂质体退火，以保持多药装载所?...

LTSL制剂是一种临床相关的脂质体产品，已开发用于提供化疗药物多索鲁比辛（DOX），并允许在临床上达到的轻度高热（T = 41°C）1高效突发药物释放。LTSL配方包括1，2-二甲酰-sn-甘油-3-磷胆碱（DPPC），脂质脂1-stearoyl-2-羟基-sn-甘油-3-磷脂酰胆碱（MSPC;M 代表"单体"） 和 PEGylates 脂质 1，2-二聚氨酯-sn-甘油-3-磷乙醇胺-N-甲氧（聚乙烯乙二醇）-2000+ （DSPE-PEG2000）。达到相变温度（Tm = 41 °C）时，脂质脂和DSPE-PEG2000一起促进膜孔的形成，导致药物2的爆裂释放。LTSL的制备主要采用批量自上而下的方法，即脂质膜水化和挤出。重新制备具有相同特性和足够数量的大批量用于临床应用仍然具有挑战性3。
微流体学是一种新兴的制备脂体技术，提供可调纳米颗粒大小、可重现性和可扩展性3。一旦优化制造参数，吞吐量可以通过并行化进行扩展，其性能与台秤3、4、5中准备的属性相同。微流体与传统的散装技术相比，一个主要优势是能够处理小液体体积，在空间和时间上具有高可控性，通过小型化，允许更快的优化，同时以连续和自动化的方式运行6。使用微流体装置生产脂质体是通过自下而上的纳米沉淀方法实现的，这种方法更具有时间和能效，因为同质化过程（如挤出和声波）是不必要的。通常，脂质的有机溶液（如乙醇）与混合无溶剂（如水和亲水有效载荷）混合。当有机溶剂与非溶剂混合时，脂质的溶解度降低。脂质浓度最终达到临界浓度，在沉淀过程触发7。脂质的纳米沉淀物最终成种，并接近于脂质体。决定脂质体大小和均匀性的主要因素是非溶剂体与溶剂之间的比率（即水与有机流动率;FRR）和溶剂环境的均匀性，在脂质自组装成脂质体8。
因此，微流体中的高效流体混合对于制备同质脂质体至关重要，混合器的各种设计已应用于不同的应用9。交错的刺骨微混频器 （SHM） 代表新一代无源混合器之一，可实现高通量（在 mL/min 范围内），具有低稀释系数。这优于传统的微流体流体动力混合装置8，10。SHM已经图案的刺骨槽，它迅速混合流体通过混沌对流9，11。SHM 的短混合时间刻度（< 5 ms，小于 10–100 ms 的典型聚合时间刻度）允许脂质自组装发生在均匀的溶剂环境中，产生尺寸均匀分布为3、12的纳米颗粒。
然而，与传统的脂质体制剂相比，由于胆固醇8缺乏，LTSL的制备并不简单，没有胆固醇8，脂质二层就容易受到乙醇引起的三位一体13、14、15的相互作用的影响。到目前为止，残留乙醇在脂质体微流体生产过程中的影响还没有得到很好的理解。大多数报告的配方本质上对间念（含胆固醇或不饱和脂质）16具有抵抗力，这与LTSL不同，它们既饱和又不含胆固醇。
此处介绍的协议使用 SHM 为温度触发释放药物输送准备 LTSL。在所提出的方法中，我们通过动态光散射（DLS）确保微流体制备的LTLS是纳米尺寸（100nm）和均匀（分散<0.2）。此外，我们使用跨膜硫酸铵梯度法（也称为远程装载）17封装了 DOX，以验证 LTSL 脂质双层体的完整性。DOX 的远程加载需要脂质体保持 pH 梯度，以实现高封装效率 （EE），如果没有完整的脂质双层，这种情况不太可能发生。在这种方法中，与典型的微流体脂质体制备方案不同，在去除乙醇之前，需要退火步骤，以实现远程加载能力;即恢复脂质双层的完整性。
如前所述，在形成LTSL期间同时封装有效载荷的初始解决方案中，也可以引入亲水和疏水性有效载荷。作为概念验证，一种FDA批准的近红外荧光染料（也是一种有前途的光热剂）被引入到初始脂质混合物中，并成功共同加载到LTSLs中。808 nm 激光器用于照射 DOX/ICG 加载 LTSL，并在 5 分钟内成功诱导多氟辛克光热触发的 DOX 爆发释放。
所有仪器和材料均可上市，即使用即用，无需定制。由于配制LTSL的所有参数都进行了优化，按照此协议，对微流体没有事先了解的研究人员也可以准备LTSL，作为热敏药物输送系统的基础。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td>Lipoid</td>
			<td>PC 16:0/16:0 (DPPC)</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000)</td>
			<td>Lipoid</td>
			<td>PE 18:0/18:0-PEG 2000 
			(MPEG 2000-DSPE)</td>
			<td></td>
		</tr>
		<tr>
			<td>1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC)</td>
			<td>Avanti Polar Lipid</td>
			<td>855775P-500MG</td>
			<td>Distributed by Sigma-Adrich; also known as Lyso 16:0 PC 
			(Not to be confused with 14:0/18:0 PC, which is also termed MSPC)</td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapters, Female Luer Lock to 1/4&#34;-28UNF</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-624</td>
			<td>Requires 2 units. For the inlets</td>
		</tr>
		<tr>
			<td>Adapters, Union Assembly, 1/4&#34;-28UNF</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-630</td>
			<td>Requires 2 units. (One unit included 2 nuts and 2 ferrules)</td>
		</tr>
		<tr>
			<td>Ammonium Sulfate ((NH4)2SO4)</td>
			<td>Sigma-Aldrich</td>
			<td>31119-1KG-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bijou vial</td>
			<td>VWR</td>
			<td>216-0980</td>
			<td>7 mL, clear, polystyrene vial</td>
		</tr>
		<tr>
			<td>Centrifugal Filter Unit</td>
			<td>Sigma-Aldrich</td>
			<td>UFC801008</td>
			<td>10 kDa MWCO, Amicon Ultra-4 Centrifugal Filter Unit</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>Heraeus Megafuge 8R</td>
			<td>With HIGHConic III Fixed Angle Rotor</td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Fisher Scientific</td>
			<td>11602609</td>
			<td>Disposable polystyrene cuvette, low volume, for DLS measurement</td>
		</tr>
		<tr>
			<td>Dialysis Kit - Pur-A-Lyzer Maxi</td>
			<td>Sigma-Aldrich</td>
			<td>PURX12015-1KT</td>
			<td>12-14 kDa MWCO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>34943-1L-M</td>
			<td></td>
		</tr>
		<tr>
			<td>DLS Instrument</td>
			<td>Malvern Panalytical</td>
			<td>Zetasizer Nano ZS90</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin Hydrochloride (DOX)</td>
			<td>Apollo Scientific</td>
			<td>BID0120</td>
			<td></td>
		</tr>
		<tr>
			<td>DSC Instrument</td>
			<td>TA Instruments</td>
			<td>TA Q200 DSC</td>
			<td></td>
		</tr>
		<tr>
			<td>DSC Tzero Hermetic Lids</td>
			<td>TA Instruments</td>
			<td>901684.901</td>
			<td>For DSC measurement</td>
		</tr>
		<tr>
			<td>DSC Tzero Pans</td>
			<td>TA Instruments</td>
			<td>901683.901</td>
			<td>For DSC measurement</td>
		</tr>
		<tr>
			<td>DSC Tzero Sample Press Kit</td>
			<td>TA Instruments</td>
			<td>901600.901</td>
			<td>For DSC measurement</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>20821.330</td>
			<td>Absolute, &#8805;99.8%</td>
		</tr>
		<tr>
			<td>FC-808 Fibre Coupled Laser System</td>
			<td>CNI Optoelectronics Tech</td>
			<td>FC-808-8W-181315</td>
			<td>FOC-01-B Fiber Collimator included.</td>
		</tr>
		<tr>
			<td>Ferrule, 1/4&#34;-28UNF to 1/16&#34; OD</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-200</td>
			<td>For the outlet</td>
		</tr>
		<tr>
			<td>Fibre Optic Temperature Probe</td>
			<td>Osensa</td>
			<td>PRB-G40</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Staggered Herringbone Micromixer (SHM)</td>
			<td>Darwin Microfluidics</td>
			<td>Herringbone Mixer - Glass Chip</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Tape</td>
			<td>Omega</td>
			<td>DHT052020LD</td>
			<td>Can be replaced by other syringe heater such as &#34;HTC&#34; or &#34;SRT series&#34; for slower heating. Manual wiring to a 3-pin plug required for 240V models</td>
		</tr>
		<tr>
			<td>Indocyanine Green</td>
			<td>Adooq</td>
			<td>A10473-100</td>
			<td>Distributed by Bioquote Limited (U.K.)</td>
		</tr>
		<tr>
			<td>Luer-lock Syringe, 5 mL</td>
			<td>VWR</td>
			<td>613-2043</td>
			<td>Hanke Sass Wolf SOFT-JECT 3-piece syringes, O.D. 12.45 mm</td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>BMG Labtech</td>
			<td>FLUOstar Omega</td>
			<td>Installed with 485 nm (exictation) and 590 nm (emission) filters</td>
		</tr>
		<tr>
			<td>Microplate, 96-well, Black, Flat-bottom</td>
			<td>ThermoFisher Scientific</td>
			<td>611F96BK</td>
			<td>For fluorescence measurement in microplate reader</td>
		</tr>
		<tr>
			<td>Microplate, 96-well, Clear, Flat-bottom</td>
			<td>Grenier</td>
			<td>655101</td>
			<td>For absorbance measurement microplate reader</td>
		</tr>
		<tr>
			<td>Nut, 1/4&#34;-28UNF to 1/16&#34; OD</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-245</td>
			<td>For the outlet</td>
		</tr>
		<tr>
			<td>PC to Pump Network Cable for Aladdin, 7ft</td>
			<td>World Precision Instruments</td>
			<td>NE-PC7</td>
			<td>Optional: Syringe pumps can be operated manually</td>
		</tr>
		<tr>
			<td>Pump control software - SyringePumpPro Software License for 2</td>
			<td>World Precision Instruments</td>
			<td>SYRINGE-PUMP-PRO-02</td>
			<td>Optional: Syringe pumps can be operated manually</td>
		</tr>
		<tr>
			<td>Pump to Pump Network Cable for Aladdin, 7 ft</td>
			<td>World Precision Instruments</td>
			<td>NE-NET7</td>
			<td>Optional: Syringe pumps can be operated manually</td>
		</tr>
		<tr>
			<td>Size exclusion chromatography (SEC) column</td>
			<td>GE Life Science</td>
			<td>17085101</td>
			<td>Sephadex G-25 resin in PD-10 Desalting Columns</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>31434-1KG-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pumps &#38; Cable (DUAL-PUMP-NE-1000)</td>
			<td>World Precision Instruments</td>
			<td>ALADDIN2-220/AL1000-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostat Temperature Controller</td>
			<td>Inkbird</td>
			<td>ITC-308</td>
			<td>Can be replaced by other syringe heater kit/thermostat</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing, ETFE (1/16&#34; OD)</td>
			<td>IDEX Health &#38; Science</td>
			<td>1516</td>
			<td></td>
		</tr>
		<tr>
			<td>USB To RS-232 Converter</td>
			<td>World Precision Instruments</td>
			<td>CBL-USB-232</td>
			<td>Optional: For computer without RS-232 port</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Grant Instruments Ltd.</td>
			<td>JB Nova 12</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic production of lysolipid-containing temperature-sensitive liposomes

所提出的协议支持高通量连续制备低温敏感脂质体（LTSLs），能够加载化疗药物，如多索鲁比辛（DOX）。为此，将乙醇脂质混合物和硫酸铵溶液注射到交错的刺骨微混剂（SHM）微流体装置中。SHM 可快速混合该解决方案，为脂质体自组装提供均匀的溶剂环境。收集的脂体首先退火，然后透析去除残留的乙醇。通过使用尺寸排除色谱，通过外部溶液的缓冲液交换，建立了硫酸铵 pH 梯度。然后，DOX 以高封装效率（> 80%）远程加载到脂体中。获得的脂质体大小均匀，Z平均直径为100nm。它们能够在轻度高热（42°C）的情况下，通过温度触发的封装DOX的突发释放。Indocyanine 绿色 （ICG） 也可以共同加载到脂质体中，用于近红外激光触发 DOX 释放。微流体方法可确保 LTSL 的高通量、可重复性和可扩展的制备。

微流体制备LTSL需要DPPC/MSPC/DSPE-PEG2000的脂质组成（80/10/10，摩尔比;LTSL10）。图 7A（左图）显示了步骤 2.9 中已制备的 LTSL10 作为透明且非粘性液体的外观。LTSL10配方是从传统配方LTSL4（DPPC/MSPC/DSPE-PEG 2000，86/10/4，摩尔比）开发的，因为LTSL4形成凝胶状粘性样品，如样品中捕获的大量气泡所示（图7...

Watch this Scientific Journal Video about Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes at JoVE.com

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

该协议提出了使用交错的刺骨微流体器装置制备热敏脂质体的优化参数。这还允许将多索鲁比辛和丁氨酸绿色共同封装到脂质体中，并产生光热触发的多索鲁比素释放，用于控制/触发药物释放。

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 ...

微流体是制备可调谐纳米粒子大小的脂质体，具有极大的可重复性和可伸缩性的新技术。该协议通过生产低温敏感脂质体，与多索鲁比辛等化疗药物和荧光染料（如丁二醇绿色）共同加载，实现连续高含量。细胞制备主要使用自上而下的方法，如脂质膜水化和挤出。为临床应用准备大量可重复的批次仍然具有挑战性。微流体的一个主要优点是能够处理小液体体积，在空间和时间上具有高可控性，同时以连续和自动化的方式运行。与张光龙一起演示手术的将是马广龙和阿玛利亚·鲁伊斯，我实验室的博士后研究人员。要组装注射器泵，请使用泵到泵网络电缆将辅助注射器泵的计算机端口连接到主注射器泵的网络端口。使用 PC 泵送网络电缆将主泵的计算机端口连接到计算机的 RS-232 串行端口。要组装雄鹿鱼骨微流体装置，请使用螺母和铁杉将管子连接到设备的每个入口和插座。然后使用第二个螺母和铁杉和联合体将两个入口的管端子转换为母耳。要设置泵控制软件，请使用注射器泵的设置按钮将主注射器泵和辅助注射器泵的地址分别分配给 Ad：01 和 Ad：02，然后在计算机上打开泵控制软件。应自动检测两个注射器泵，然后发出蜂鸣声。选择 HSW Norm-Ject 5cc 直径等于 12.45，将直径分配给 12.45 毫米。将泵 1 的速率设置为每分钟 0.25 毫升，泵 2 的速率设置为每分钟 0.75 毫升。将音量设置为超过五毫升的任何值，并选择两个泵的输液模式。然后单击"设置"以确认设置。要准备 LTSL10 或 LTSL10 ICG 脂质混合物，请使用一个五毫升的 luer 锁注射器提取一毫升脂质混合物和另一个五毫升的 luer 锁注射器，以提取至少三毫升硫酸铵溶液。将注射器的筒法兰滑到泵的注射器固定器上，将加载的注射器安装到注射器泵上，将注射器的柱塞法兰连接到泵的推杆块上，将加热胶带的另一端和温度探头与带恒温器的注射器周围，并附着含有脂质溶液的注射器周围的温度探头。将加热胶带的端端包裹到含有水溶液的注射器上，将含有脂质混合物的注射器连接到乙醇入口，然后将含有硫酸铵溶液的注射器连接到水性入口。根据需要调整柱塞位置，以清除注射器中的任何气泡，并每隔 10 秒插入并拔下加热胶带，直到注射器的温度达到约 51 度。当恒温器显示适当的温度时，单击泵控制软件中运行所有泵以运行注射器泵，并检查液流量是否无气泡和任何泄漏。将脂蛋白酶样本收集到小瓶中，当任何一个注射器几乎是空的时，暂停或停止输液。将收集的脂体溶液在60摄氏度的水浴中冲洗1-1~2小时，然后将溶液转移到透析管中透析，对一升240毫摩尔硫酸铵进行透析，在37摄氏度下至少4小时。然后将纯化的脂质体储存在摄氏四度。对于通过跨膜 pH 梯度对脂质体进行远程加载，将 25 毫升 HBS 加载到大小排除色谱柱的顶部，并允许所有 Eluent 流经柱。将一毫升透析脂质体加载到柱上。当所有脂质溶液都通过该列时，向列中加入 1.5 毫升的新鲜 HBS。当所有缓冲区都通过柱时，向列中添加三毫升新鲜 HBS 并收集吸水器。接下来，在Biju小瓶中，以1：20 DOX：磷脂摩尔比添加 DOX 溶液，将缓冲液交换脂质体溶液加入一毫升，并将小瓶放在37摄氏度的水浴中1-1~2小时。装料后，通过大小排除色谱净化脂质体，正如刚才所证明的。对于激光加热诱导的电触发释放脂体内容物，将水浴设置为37摄氏度。当温度稳定下来后，在透明96井板的每个井中加入200微升的DEX装脂体，并将板放在水浴中，底部浸入水中。然后将激光系统电流设置为 2.27 安培，将激光系统准直器垂直放置在 96 井板表面上方 5 厘米。打开激光器并使用光纤温度探头每分钟监控一次温度。在5分钟和10分钟时，从透明96孔板的每个孔中吸入10微升的DEX，并将190微升的HBS加入到黑色96孔板的三个单独孔中。为了评估完整的药物释放，将10微升脂体与170微升的HBS和20微升的1%Triton X-100洗涤剂溶液混合在黑色96孔板的三个单独井中。然后测量板读卡器上的 DOX 荧光强度。LTSL4 的微流体生产可产生凝胶状粘性溶液，如大量受困气泡所示，而 LTSL10 的制备则导致形成透明的非粘性液体。在51摄氏度下准备的LTSL10动态光散射测量表明，预计的Z平均直径和分散度表明实验成功。然而，当 LTSL10 在 20 摄氏度下准备时，获得更大、更分散的脂质体，从而产生次优产品。由传统的带挤出的脂质薄膜水化方法编写的LTSL表明，在退火时，由微流体准备的LTSL10具有50-80%的 DOX 封装效率，使 DOX 封装效率显著提高，平均达到 85%，表明 DOX 的远程加载成功，并且存在跨膜 pH 梯度。LTSL10 的 DOX 释放配置文件已确定为温度敏感。LTSL10 的相变相对较宽，发病温度为 41.6 摄氏度，峰值为 42.6 摄氏度。ICG 装载的效率取决于初始 ICG 浓度以及样品的大小和分散性。此外，LTSL10 ICG辐照与近红外激光诱导光热加热触发DEX的释放。在尝试此程序时，必须确保稳定的流体流动，使流体的混合以及脂质体的形成保持可重复性。脂体退火和透析是确保稳定的脂体配方和高载荷能力的两个重要步骤。还可以进行体内测试，以评估 LTSL10 生物分布、药物释放和抗癌活性。我们的协议可用于成功微流体生产无胆固醇含糖脂含热脂质体，用于药物输送应用。

Research

JoVE Journal

Bioengineering

A Microfluidic Technique to Probe Cell Deformability

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 ...

科研

生物工程

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 ...

A Microfluidic Chip for ICPMS Sample Introduction

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

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 ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

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

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 ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...