Name: preparation, administration, and assessment of in vivo tissue-specific cellular uptake of fluorescent dye-labeled liposomes
Uploaded: 2020-07-30T14:45:00.000+00:00
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Description: Watch this Scientific Journal Video about Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes at JoVE.com

作者要感谢Michael Solga和其他流式细胞术核心工作人员提供流式细胞术培训和服务。作者还要感谢Shiva Sai Krishna Dasa，Dustin K. Bauknight，Melissa A. Marshall，James C. Garmey，Chantel McSkimming，Aditi Upadhye和Prasad Srikakulapu在脂质体制备（SSKD，DKB），组织收获（MAM，JCG）以及流式细胞术染色和样品采集（AU，PS，CM）方面的帮助。这项工作得到了阿斯利康，R01HL 136098，R01HL 141123和R01HL 148109，AHA 16PRE30770007和T32 HL007284资助的支持。

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作者没有什么可透露的。

在这里，我们描述了一个由三部分组成的方案，用于（i）制备用荧光脂质染料标记并加载有抗糖尿病化合物tesaglitazar的脂质体，（ii）通过眶后注射将脂质体施用于小鼠，以及（iii）收获，处理和染色组织以通过流式细胞术检测细胞水平的脂质体摄取。该协议回顾了大约150μm脂质体的制备和脂肪，血液和脾脏摄取的评估。脂质体制备是可扩展的，主要在室温下进行，并利用反相蒸发来最大化药物负载和有机溶剂的去除。使用该协议，纯化的脂质体样品中可以达到高达2 mg / mL的tesaglitazar浓度。制备的脂质体可以在4°C的HEPES缓冲液中储存一年以上。根据我们的经验，它们证明了平均粒径的最小变化。在用10 kDa离心过滤器超滤分离脂质体与外部药物后，通过分光光度法证明了低于10%的药物含量损失。
在脂质体制备过程中，需要考虑一些关键步骤和因素。首先，协议步骤的顺序很重要，必须遵守。其次，上样替格列塔扎时所用溶液的pH值必须保持在7.4，以最大限度地提高溶解度和有效上样量。第三，设备和过滤器的正确组装确保每个步骤的输出具有适当的尺寸和纯度。例如，如果 100 纳米和 200 纳米滤光片组装不当，则可能会导致一批异质性和尺寸不合适的脂质体。第四，需要在载药前完全去除醋酸钙，以最大限度地将替格列塔唑转移到脂质体中。要测试乙酸钙的完全去除，请使用高速沉降去除脂质体，然后测量非脂质体溶液中的乙酸钙水平。第五，重要的是在每一步称重并记录添加到脂质体制剂中的所有材料的质量。这确保了可以计算出适当的浓度并保持所需的材料比例。最后，如果技术执行不当，则可能存在不希望的异质性水平。使用DLS和其他方法（如电子显微镜）彻底检查此参数非常重要。要提高均匀性，请考虑调整所选过滤器尺寸或堆叠两个过滤器。
此外，在完全实施此方案之前，计划和优化流式细胞术的对照和抗体组合至关重要（表1， 表3）。应检测抗体，以确保使用适当的浓度进行染色，并且荧光团之间的重叠最小。脂质体制备过程中所用染料的激发和发射也必须考虑在组合规划中。在我们的结果中，我们使用了DiD，它与异藻蓝蛋白（APC）和AlexaFluor 647等荧光团具有相似的激发和发射。因此，我们没有在抗体组合中选择与这些荧光团偶联的抗体。此外，同种型对照不包括在该协议中。这是因为为该方案选择的抗体是经过充分验证的市售抗体。但是，如果有兴趣使用以前未优化的抗体，请考虑在进行完整实验之前对感兴趣的组织进行针对同种型对照的抗体测试。
虽然该协议演示了如何从小鼠治疗后提取和处理血液，脾脏，腹股沟脂肪和附睾脂肪组织，但这种通用方法可以应用于其他组织。根据感兴趣的组织，可能需要改变以下组织的处理和消化方案:肺21，肝22，腹膜腔3，骨髓3，23，脑24。
这种方法的一个重要局限性是，每只动物只能在一个时间点评估摄取。因此，将该协议与其他非侵入性成像技术相结合或相应地计划以确保有足够的资源进行评估可能是有利的。细胞摄取和细胞周转的时间是需要考虑的重要因素:脂质体将在前24小时内在全身循环，并且取决于吸收脂质体的细胞的寿命或它们对摄取的反应，可能会发生细胞死亡或进一步吞噬作用。我们之前的研究表明，DiD+ 种群在不同时间点的种群特征发生了变化3。因此，评估较早的时间点或与目的机制生物学最相关的时间点的摄取非常重要。此外，虽然可以使用该协议对整个组织中的细胞摄取进行定量，但流式细胞术不能揭示组织定位。将这种方法与组织学方法相结合有助于解决这一限制。
通常，该协议补充了现有方法，例如组织学和全身荧光成像。随着流式细胞术工具和方法的不断进步，为越来越多的特异性细胞群开发更大的组合将成为可能。我们建议在上述方法之外使用该协议，因为这将改善细胞摄取的评估，并提供验证流式细胞术观察到的结果的机会。例如，是否应该发现脂肪组织中的大部分颗粒被流式细胞术的巨噬细胞吸收。可以保存，固定，切片和染色相同脂肪组织的额外等分试样的免疫荧光以获取巨噬细胞标志物，以验证细胞类型确实吸收脂质体。这种方法应该增加纳米颗粒生物分布测定的严谨性:验证细胞特异性靶向，量化细胞摄取，识别脱靶摄取，并希望提供信息以产生观察到的治疗结果的机制假设。该协议也可以适用于使用不同脂质体的未来研究，研究其他组织中的摄取，以及在肥胖和代谢障碍或任何其他疾病的情况下测试新化合物，其中纳米颗粒递送是一种可行的治疗选择。

随着对开发利用纳米颗粒药物递送的疗法的兴趣不断增长，制备和评估颗粒分布和摄取的方法必须继续推进，扩展，并为研究界所用1，2。该协议旨在评估在用装有tesaglitazar（一种过氧化物酶体增殖物激活受体（PPAR）-α/γ激动剂3，4）的DiD标记脂质体处理后在体内吸收脂质体的确切细胞类型。在这些研究中，我们能够评估脂质体tesaglitazar治疗直接影响哪些细胞类型，靶向部分的功效，并生成假设来解释我们观察到的治疗结果。此外，在多种细胞类型中建立的生物学功能表明吞噬细胞如巨噬细胞、树突状细胞和肝脏特异性库普弗细胞占据了大部分脂质体5，6，7。利用该协议，我们已经证明非经典吞噬细胞也可以吸收脂质体3，4。
该协议提供了一种优化的方法，用于溶解tesaglitazar，通过反相蒸发制备脂质体，并使用醋酸钙作为远程药物加载的引诱剂。所提供的方法可供许多实验室使用，并且缺乏难以获得的材料和需要高温的步骤。该方案产生大小的脂质体，最适合增加体内循环8。此外，正如Su等人所总结的那样，迄今为止，已经对评估体内脂质体分布和组织摄取的方法进行了深入研究和测试9。正电子发射断层扫描（PET），磁共振成像（MRI）和荧光分子断层扫描（FMT）方法用于量化组织特异性生物分布和摄取9，10，11。虽然这些方法已经过优化以最大限度地提高体内检测率，但它们仍然缺乏以细胞分辨率量化体内脂质体摄取的能力。这里介绍的方案旨在通过使用流式细胞术来满足这一需求。最后，对于该协议，细胞摄取缩小到包括脂肪组织在内的一些组织。越来越多的文献调查了在肥胖，代谢障碍和炎症的情况下使用纳米颗粒提供治疗的潜力12，13，14，15，16，17。因此，我们认为与处理和分析脂肪组织的有效方法共享协议很重要 - 脂肪组织是在这些病理中起重要作用的组织之一。

<table><tbody><tr><td>1-mL syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10-mL syringe</td><td>BD</td><td>302995</td><td></td></tr><tr><td>25-gauge needle, sterile for retro-orbital injection</td><td>BD</td><td>305122</td><td></td></tr><tr><td>27-gauge needle, sterile for retro-orbital injection</td><td>BD</td><td>305620</td><td></td></tr><tr><td>Anti-mouse B220 BV421</td><td>Biolegend</td><td>103251</td><td>Clone RA3-6B2</td></tr><tr><td>Anti-mouse CD115 PE</td><td>eBioscience</td><td>12-1152-82</td><td>Clone AFS98</td></tr><tr><td>Anti-mouse CD11b PerCP Cy5.5 antibody</td><td>BD Biosciences</td><td>550993</td><td>Clone M1/70</td></tr><tr><td>Anti-mouse CD11c APC ef780 antibody</td><td>eBioscience</td><td>47-0114-82</td><td>Clone N418</td></tr><tr><td>Anti-mouse CD19 PE CF594</td><td>BD Biosciences</td><td>562291</td><td>Clone 1D3</td></tr><tr><td>Anti-mouse CD3 FITC antibody</td><td>BD Biosciences</td><td>553061</td><td>Clone 145-2C11</td></tr><tr><td>Anti-mouse CD31 BV605</td><td>Biolegend</td><td>102427</td><td>Clone 390</td></tr><tr><td>Anti-mouse CD45 PerCP</td><td>BD Biosciences</td><td>557235</td><td>Clone 30-F11</td></tr><tr><td>Anti-mouse F4/80 PE Cy7</td><td>Biolegend</td><td>123114</td><td>Clone BM8</td></tr><tr><td>Bovine serum albumin</td><td>Gemini Bio-products</td><td>700-107P</td><td></td></tr><tr><td>Desalting spin-column</td><td>ThermoFisher</td><td>89889, 89890</td><td>Zeba spin column</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Dynamic Light Scattering, Nicomp 370</td><td>Particle Sizing System, Inc</td><td></td><td></td></tr><tr><td>FIX &amp;amp; PERM Cell Permeabilization Kit</td><td>ThermoFisher Scientific</td><td>GAS004</td><td></td></tr><tr><td>Gauze sponges</td><td>Dermacea</td><td>441211</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>3393-1MU</td><td></td></tr><tr><td>Liposome extruder</td><td>Millipore Sigma</td><td>Z373400</td><td>LiposoFast</td></tr><tr><td>Live/Dead Aqua</td><td>ThermoFisher Scientific</td><td>L34957</td><td></td></tr><tr><td>Nanosight</td><td>Malvern Instruments Ltd</td><td>NS300</td><td></td></tr><tr><td>Ophthalmic lubricant</td><td>Optixcare</td><td></td><td>20g/70 oz Sterile</td></tr><tr><td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td><td>Alfa Aesar</td><td>433689L</td><td></td></tr><tr><td>Polyethylene vial for mincing</td><td>Wheaton</td><td>986701</td><td></td></tr><tr><td>Rotary evaporator</td><td>Buchi</td><td>Re111</td><td></td></tr><tr><td>Sonicator</td><td>Misonix</td><td>XL2020</td><td></td></tr><tr><td>T/Pump Heat therapy pump and pad</td><td>Gaymer Industries</td><td>TP-500</td><td></td></tr><tr><td>Tesaglitazar</td><td>Tocris</td><td>3965</td><td></td></tr><tr><td>Track-etched polycarbonate membranes</td><td>Thomas Scientific</td><td>1141Z**</td><td>Whatman, Nuclepore Polycarbonate hydrophilic membranes</td></tr><tr><td>ZetaSizer/DLS-ELS system</td><td>Malvern Instruments Ltd</td><td></td><td></td></tr></tbody></table>

preparation, administration, and assessment of in vivo tissue-specific cellular uptake of fluorescent dye-labeled liposomes

人们对使用脂质体在体内递送化合物的兴趣日益浓厚，特别是对于靶向治疗方法。根据脂质体配方，脂质体可以优先被体内不同的细胞类型吸收。这可能会影响治疗颗粒的疗效，因为不同疾病的进展是组织和细胞类型特异性的。在该协议中，我们提出了一种使用DSPC，胆固醇和PEG-2000 DSPE以及脂质染料DiD作为荧光标记合成和荧光标记脂质体的方法。该协议还提供了一种在体内递送脂质体和使用流式细胞术评估脂质体的细胞特异性摄取的方法。这种方法可用于确定吸收脂质体的细胞类型，并量化脂质体摄取在细胞类型和组织中的分布和比例。虽然本协议中未提及，但额外的测定（例如细胞仪上的免疫荧光和单细胞荧光成像）将加强所做的任何发现或结论，因为它们允许评估细胞内染色。方案可能还需要根据感兴趣的组织进行调整。

脂质体生产
这里发表的结果与我们之前发表的工作3，4，20中的结果相似。利用此处介绍的方案，我们期望产生大小约为150-150-160 nm的脂质体。DLS 显示平均脂质体直径为 163.2 nm，zeta 电位为 -19.2 mV（图 1A）。低温电子显微镜（cryo-EM）成像显示环状脂质体（图1B），DLS图显示与平均直径的标准偏差相对较小（图1C）。
脂质体结合阳性需要 PBS 处理的对照
我们小组先前使用该协议的研究调查了体内给药一周后脂肪SVF，脾脏和血液中与脂质体结合的细胞亚群3，4。使用PBS处理的小鼠，用脂质体处理小鼠样品上使用的相同抗体组合对腹膜腔和脾脏细胞进行染色。治疗一周后收获组织（图2A）。来自PBS处理的小鼠的样品用作DiD FMO，用于创建阳性DiD门（图2B，C）。可以使用DiD正信号创建正栅极，但缺少DiD信号的样品也必须用于验证正栅不包含任何DiD阴性样本。
需要滴定以优化荧光信号
在进行完整实验之前，必须优化各种条件，包括细胞染色期间使用的荧光偶联抗体的浓度和脂质体制备期间使用的脂质染料的浓度。流式细胞仪具有荧光强度的检测上限，因此脂质体中掺入的染料过多会导致通过细胞仪的样品中DiD信号水平无法量化。此外，脂质体中过多的DiD可能导致高水平的非特异性染料转移，这可能会扭曲细胞摄取结果。图3报告了一项实验的结果，在该实验中，滴定脂质染料的浓度以确定在使用流式细胞仪检测范围内产生最佳信号的浓度。这是在最终实验感兴趣的组织上进行的:血液（图3A），腹股沟脂肪SVF（图3B）和附睾脂肪SVF（图3C）。选择用于测试的浓度为每1 mL脂质体10mg DiD（高，红色），1mg DiD（中，蓝色）或0.1mg DiD（低，灰色）。脂质体中使用的最高浓度过高，在所有三种组织中都超过了细胞仪的可定量范围（图3A\u2012C，红色）。最低浓度的DiD显示出一些信号（图3 A\u2012C，灰色），但没有观察到PBS处理的细胞之外的清晰群体（图3A\u2012C，黑色）。量化时，每种组织和浓度的DiD MFI的算术平均值表明PBS对照与DiD的中等浓度之间存在明显区别（图3D）。因此，如协议所示，我们选择了中间浓度（图3，蓝色）用于我们的脂质体制备。
使用多抗体组合可以鉴定不同细胞亚群对脂质体的摄取
使用表3中概述的面板，用针对巨噬细胞，B细胞，T细胞，树突状细胞，单核细胞和内皮细胞标记物的抗体对细胞进行染色（图4）。每种组织类型需要略有不同的门控策略，但可以在每种组织中鉴定出大多数相同的细胞类型。一些例外包括内皮细胞，这通常不在血液中发现，以及单核细胞，它们在血液中的频率通常高于其他组织。一旦确定了群体，就可以量化每个细胞群的总大小及其DiD +的频率。可以进行进一步的计算来表征DiD +群体:DiD +细胞中巨噬细胞，内皮细胞等的百分比是多少。请注意，这些是示例门控策略，但不是分析样本的唯一方法。分析将由所选的试剂盒和可用的流式细胞仪决定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61585/61585fig01.jpg"> 图1:制备的脂质体的示例特征。 （A）如上所述测量大小和zeta电位，并以表格形式报告。每个参数都表示为平均值±标准差。（B）冷冻电镜用于对制备的脂质体进行成像。白色比例尺的长度为 50 nm。（C）在该制备中，使用DLS生成脂质体直径的直方图。该图改编自Osinski等人3。<a href="https://www.jove.com/files/ftp_upload/61585/61585fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61585/61585fig02.jpg"> 图2:来自PBS或脂质体处理小鼠的代表性DiD染色。 （A）PBS和脂质体处理的实验示意图。PBS或脂质体在一周内注射三次。在治疗的第8天收获组织。（乙、丙）代表性流图显示脂质体处理的（C）小鼠的DiD染色阳性，但未显示PBS处理的（B）小鼠的DiD染色阳性。FSC，前向散射。 <a href="https://www.jove.com/files/ftp_upload/61585/61585fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61585/61585fig03.jpg"> 图 3:脂质体中 DiD 的滴定。 用三种不同浓度的DiD制备脂质体并注射到小鼠体内。灰色表示每 1 mL 脂质体 0.1 mg DiD 的低浓度，蓝色表示 1 mg DiD/mL 脂质体的中等浓度，红色表示 10 mg DiD/mL 脂质体的高浓度。使用PBS处理的小鼠作为阴性对照（黑色）。注射后24小时收获血液（A，圆形），腹股沟脂肪（B，三角形）和附睾脂肪（C，方形），并进行处理以分离单细胞悬液。这些样品在流式细胞仪上运行至可检测的DiD水平。呈现每个治疗组叠加的组织特异性直方图，以显示每个浓度的荧光强度（A\u2012C）。还量化了每种组织和浓度的DiD的算术平均值，并绘制了（D）。SSC = 侧向散射。 <a href="https://www.jove.com/files/ftp_upload/61585/61585fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61585/61585fig04v4.jpg"> 图 4:脂肪 SVF、血液和脾脏中细胞亚群的代表性流式细胞术分析。 （A\u2012C）用于识别脂肪SVF（A），脾脏（B）和血液（C）中的细胞亚群和DiD +细胞的门控策略的示意图。缩写:FSC = 前向散射;LD = 活/死;L-DC = 淋巴样树突状细胞;M-DC = 髓系树突状细胞;SSC = 侧向散射。该图改编自Osinski等人3。<a href="https://www.jove.com/files/ftp_upload/61585/61585fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>控制</td>
			<td>目的</td>
		</tr><tr><td rowspan="5">用PBS或生理盐水处理的小鼠</td>
			<td>使用该小鼠的细胞进行以式细胞术对照:</td>
		</tr><tr><td>1. 未染色细胞</td>
		</tr><tr><td>2. 活/死单污渍</td>
		</tr><tr><td>3. 用全面板染色的细胞，但缺乏脂质体荧光，无法在分析过程中确定阳性脂质体信号</td>
		</tr><tr><td>这/这些小鼠/小鼠也将用于确定脂质体在体内是否有任何影响，因为您将在实验中具有非脂质体对照。</td>
		</tr><tr><td>卸载的脂质体</td>
			<td>如果您在脂质体中加载化合物，则应在没有该化合物的情况下合成部分脂质体批次。这解释了脂质体单独对体内的任何影响。</td>
		</tr><tr><td>单独使用迪德</td>
			<td>由于DiD也可以被细胞膜吸收，因此分配一些小鼠以等于脂质体中发现的量接受游离染料将有助于解释任何背景膜染色。</td>
		</tr><tr><td>荧光负一 （FMO） 控制</td>
			<td>这些细胞被面板中除一种抗体以外的所有抗体染色。与上面框中的#3一样，这有助于在分析过程中确定该抗体的真阳性信号</td>
		</tr></tbody></table>表 1:要在此协议中使用的控件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>溶液</td>
			<td>组件</td>
			<td>每批/小鼠所需的近似体积</td>
		</tr><tr><td colspan="3">脂质体制备</td>
		</tr><tr><td>醋酸钙</td>
			<td>1 M 乙酸钙在 H2O</td>
			<td>50毫升</td>
		</tr><tr><td>HEPES缓冲液</td>
			<td>10 mM HEPES H2O，pH 7.4</td>
			<td>50毫升</td>
		</tr><tr><td>赫佩斯的特萨格利塔扎</td>
			<td>在 10 mM HEPES 中</td>
			<td>10毫升</td>
		</tr><tr><td colspan="3">组织采集、处理和染色</td>
		</tr><tr><td>磷酸盐缓冲溶液</td>
			<td>137 mM 氯化钠、2.7 mM 氯化钾、10 mM Na 2 HPO 4、1.8 mM KH 2 PO4 蒸馏 H 2O溶液</td>
			<td>2毫升</td>
		</tr><tr><td>PBS-肝素</td>
			<td>0.1 mM 肝素在 PBS 中</td>
			<td>10毫升</td>
		</tr><tr><td>HEPES缓冲液</td>
			<td>20 毫米丙二醇中的 HEPES</td>
			<td>5毫升</td>
		</tr><tr><td>消化缓冲液</td>
			<td>HEPES 缓冲液中的 2 mg/mL I 型胶原酶</td>
			<td>5毫升</td>
		</tr><tr><td>AKC裂解缓冲液</td>
			<td>0.158 米 NH3厘升， 10 毫米氢氧化钾3， 0.1 毫米钠 2 EDTA 在 ddH 2 O， pH7.2中</td>
			<td>15毫升</td>
		</tr><tr><td>FACS 缓冲液</td>
			<td>1% BSA， 0.05% NaN3 在 PBS 中</td>
			<td>15毫升</td>
		</tr><tr><td>Fc 块（稀释）</td>
			<td>FACS 缓冲液中的 1:50 Fc 块</td>
			<td>250 微升</td>
		</tr><tr><td>固定缓冲液</td>
			<td>PBS中2%多聚甲醛</td>
			<td>200 微升</td>
		</tr></tbody></table>表 2:要准备的解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>一个</td>
			<td>B</td>
			<td>C</td>
			<td>D</td>
		</tr><tr><td colspan="4">细胞外染色（2x 抗体混合物）</td>
		</tr><tr><td>抗原</td>
			<td>荧光基团</td>
			<td>每 100 μL 测试的 Ab 体积</td>
			<td>所需总量:</td>
		</tr><tr><td>CD45</td>
			<td>全氯乙烯</td>
			<td>0.5 微升</td>
			<td>C 列 x 1.2 x 总计 # 个样本</td>
		</tr><tr><td>CD11b</td>
			<td>全氯乙烯5.5</td>
			<td>0.25 微升</td>
			<td>（0.5 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>F4/80</td>
			<td>PE Cy7</td>
			<td>0.25 微升</td>
			<td>（0.25 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>CD19</td>
			<td>聚乙烯-CF594</td>
			<td>1 微升</td>
			<td>（0.25 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>CD3</td>
			<td>FITC</td>
			<td>1 微升</td>
			<td>（1.0 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>CD31</td>
			<td>BV605</td>
			<td>0.25 微升</td>
			<td>等。。。</td>
		</tr><tr><td>CD11c</td>
			<td>APC ef780</td>
			<td>1 微升</td>
			<td></td>
		</tr><tr><td>CD115</td>
			<td>体育</td>
			<td>1.5 微升</td>
			<td></td>
		</tr><tr><td colspan="4">要创建抗体混合物，请将 D 列中计算的抗体与 FACS 缓冲液或亮紫染色缓冲液*合并至最终体积（50 μL x 1.2 x 总 # 样品）</td>
		</tr><tr><td colspan="4">活/死染色 （1x）</td>
		</tr><tr><td>生/死</td>
			<td>荧光基团</td>
			<td>每 200 uL 测试的 L/D 体积</td>
			<td>所需总量:</td>
		</tr><tr><td>生/死</td>
			<td>水</td>
			<td>0.67 微升</td>
			<td>C 列 x 1.2 x 总计 # 个样本</td>
		</tr><tr><td colspan="4">细胞内染色 （1x）</td>
		</tr><tr><td>抗原</td>
			<td>荧光基团</td>
			<td>每 50 μL 测试的 Ab 体积</td>
			<td>所需总量:</td>
		</tr><tr><td>αSMA</td>
			<td>FITC</td>
			<td>0.125</td>
			<td>C 列 x 1.2 x 总计 # 个样本</td>
		</tr><tr><td colspan="4">*如果您的组合中使用了多种与亮紫荧光团偶联的抗体，则应使用亮紫染色缓冲液。</td>
		</tr></tbody></table>表 3:用于流式染色的示例抗体组合和染色混合物的计算。

Watch this Scientific Journal Video about Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes at JoVE.com

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

该协议的目标是合成荧光标记的脂质体，并使用流式细胞术在细胞水平上鉴定脂质体的体内定位。

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 ...

preparation-administration-assessment-vivo-tissue-specific-cellular

Research

JoVE Journal

Bioengineering

4.1K 次观看.  University of Virginia. 该协议的目标是合成荧光标记的脂质体，并使用流式细胞术在细胞水平上鉴定脂质体的体内定位。

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

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

科研

癌症研究

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

发育生物学

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

生物工程

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

灌注的空间测量，实体肿瘤间质流体压力和脂质体积累

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

医学

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

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

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 ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

指某东西的用途<em&gt;离体</em&gt;全器官成像和定量组织组织学确定荧光标记分子的生物分布

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

生物学

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest in developing these peptides, especially cell-specific ones, as novel vectors for delivering diagnostic and therapeutic materials. Our past work involving phage display identified a novel, nonnaturally occurring, 12 amino acid-long peptide that we named cardiac targeting peptide (CTP) due to its ability to transduce normal heart tissue in vivo with peak uptake seen in as little as 15 min after an intravenous injection. We have undertaken detailed biodistribution studies by injecting CTP labeled with fluorophore cyanine5.5, allowing it to circulate for various periods of time, and euthanizing, fixing, and sectioning multiple organs followed by fluorescent microscopy imaging. In this publication, we describe these processes as well as ex vivo imaging of harvested organs using an in vivo imaging system in detail. We provide detailed methodologies and practices for undertaking transduction as well as biodistribution studies using CTP as an example.

We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.

心脏靶向肽转导效率的活体成像

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest ...

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

摘要

探索更多视频

此视频中的章节

综合程序，以评估

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

灌注的空间测量，实体肿瘤间质流体压力和脂质体积累

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

使用体外活体细胞成像技术研究脂纳米粒子化疗

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

指某东西的用途

心脏靶向肽转导效率的活体成像

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

摘要

探索更多视频

此视频中的章节

综合程序，以评估

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

灌注的空间测量，实体肿瘤间质流体压力和脂质体积累

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

使用体外活体细胞成像技术研究脂纳米粒子化疗

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

指某东西的用途

心脏靶向肽转导效率的活体成像

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像