作者要感谢Emilie Josse对手稿的批判性评论。这项工作得到了"癌症联盟"基金会、"癌症研究基金会"和"国家科学研究中心"（CNRS）的支持。

1. 大型单层囊泡（LUV）的制备
<ol><li>准备LUV，用作荧光泄漏测定的细胞膜模拟物。</li>
	<li>与汉密尔顿玻璃注射器磷脂酰胆碱（DOPC，786.11克/摩尔），鞘磷脂（SM，760.22克/摩尔）和胆固醇（Chol，386.65克/摩尔）以4:4:2的摩尔比混合。脂质溶液从在甲醇/氯仿（3/1;体积/体积）溶剂中以25mg/mL在25mL玻璃圆底烧瓶中溶解的每种脂质的储备溶液中获得。基于4μmol的DOPC，4μmol的SM和2μmol的Chol，通过分别混合126μL，117μL和31μL从储备溶液中获得脂质溶液。 注意:甲醇是一种有毒易燃溶剂，氯仿有毒且致癌。两者都应在引擎盖下进行适当的保护。</li>
	<li>在60°C下真空下使用旋转蒸发器蒸发甲醇/氯仿45-60分钟，直到形成干燥的脂膜。</li>
	<li>准备两种高效浓缩液缓冲液。通过混合20 mM HEPES（238.3 g / mol）和75 mM NaCl（58.44 g / mol）来制备HEPES缓冲液1，并将pH值调节至7.4。通过混合20 mM HEPES和145 mM NaCl来制备HEPES缓冲液2，并将pH调节至7.4。HEPES缓冲液可以在4°C下储存1个月。 注意:建议使用渗透压计检查缓冲液的渗透压。</li>
	<li>通过将膜不透性的荧光染料淬灭剂对，8-氨基萘-1，3，6-三磺酸，12.5mM（ANTS，427.33 g / mol）的二钠盐和对二甲苯 - 双吡啶溴化物以45mM（DPX，422.16 g / mol）溶解在HEPES缓冲溶液中来制备脂质水合溶液。将蚂蚁与DPX混合可得到黄色溶液。为了达到12.5mM的ANTS和45mM的DPX的浓度，分别溶解在4mLHEPES缓冲液1中21.4mg和76mg。 注意:脂质水合溶液可以通过用铝箔包裹管在4°C下储存2周。</li>
	<li>通过用1mL脂质水合溶液重悬干燥的脂质膜并通过涡旋直到干燥的脂质膜溶解来重建多层囊泡（MLV）。确保溶液完全溶解，因为小脂质聚集体会对上述步骤产生负面影响。另外，检查玻璃圆底烧瓶的壁，以确保没有残留的脂质膜。 注意:溶解后溶液会出现乳白色和淡黄色。</li>
	<li>将囊泡置于五个冷冻/解冻循环中，以获得单层囊泡。通过将玻璃圆底烧瓶在液氮中放置30秒以进行冷冻步骤，然后将其留在水浴中2分钟进行解冻步骤来执行每个循环。 注意:洗澡水的温度应比脂质的熔化温度高5-10°C。</li>
	<li>通过将两个过滤载体插入预先加湿的HEPES缓冲液在每个聚四氟乙烯（PTFE）挤出机片中置于金属挤出机罐中的固体挤出机来制备脂质挤出机。</li>
	<li>将HEPES加湿聚碳酸酯膜（孔径为0.1μm，直径25 mm）放在一个过滤器支架的顶部。</li>
	<li>组装两个金属挤出机罐并拧紧它们。</li>
	<li>将组装好的挤出机放入支架中，并在每个聚四氟乙烯挤出机片末端的适当孔中引入1 mL注射器。挤出对应于被测液体从一个注射器通过聚碳酸酯膜到另一个注射器的通道。</li>
	<li>使用装入 1 mL 注射器之一的 1 mL HEPES 缓冲液测试挤出机，以确保没有泄漏或问题。</li>
	<li>用MLV样品代替1 mL HEPES缓冲液。</li>
	<li>通过将MLV样品从一个注射器通过聚碳酸酯膜传递到另一个注射器至少21次来进行挤出，以获得相同尺寸的均匀LUV。 注意:挤出应在高于脂质混合物熔化温度的温度下进行。</li>
</ol>2. 润滑油的净化
<ol><li>准备色谱柱纯化以除去未封装的ANTS和DPX过量。</li>
	<li>将交联葡聚糖凝胶（G-50）重悬于液相色谱柱（鲁尔锁，非夹套，1.0厘米×20 厘米，床体积16毫升）中，重悬于0.01%NaN 3（65克/摩尔）的水性培养基中，直至柱无色部分顶部下方1厘米处。</li>
	<li>打开水龙头，让液体流动以沉降交联葡聚糖凝胶。</li>
	<li>通过用20 mL HEPES缓冲液2洗脱来洗涤色谱柱，并丢弃色谱柱的输出流量。</li>
	<li>一旦柱上方溶剂的死体积最小化（<100μL），但足以避免交联葡聚糖凝胶的任何干燥，请关闭水龙头。</li>
	<li>将新鲜挤出的LUV（黄色）放在色谱柱上，让它们进入交联的葡聚糖凝胶中。</li>
	<li>连续向色谱柱中加入HEPES缓冲液2以执行LUV纯化。</li>
	<li>洗脱约2 mL HEPES缓冲液2（不要忘记定期填充色谱柱顶部以避免干燥交联葡聚糖凝胶）:游离的黄色ANTS和DPX溶液的迁移速度比脂质体慢。</li>
	<li>开始在试管（1.5 mL）中收集纯化的LUV。</li>
	<li>观察柱中的淋洗液滴，当它们变成乳白色时，它们含有脂质体。更换试管以回收含LUV的馏分。</li>
	<li>洗脱，直到滴剂不再乳白色（约1毫升）。之后，在单独的馏分中洗脱另外0.5 mL，然后停止洗脱。 注意:标准品现在提供多种分子量，如试剂盒或单个分子量，用于校准LUV的洗脱体积。</li>
	<li>用LUV包裹在铝箔中的管子，以避免荧光染料漂白。</li>
	<li>用20 mL HEPES缓冲液洗涤色谱柱 2.</li>
	<li>然后，LUV可以在4°C下储存一周。 注意:由于LUV稳定性可能取决于LUV浓度和组成以及离子强度，因此应使用动态光散射（DLS）仪器控制LUV的大小（见第4节）。在每次测试之前表征LUV尺寸和均匀性）。</li>
</ol>3. 量化LUV的浓度
<ol><li>通过磷脂定量试剂盒估计LUV浓度，从而可以评估胆碱浓度19。当含有胆碱的磷脂含量很大（>50%的LUV）时，可以应用该测定。</li>
	<li>通过将18mg染色原底物溶解在提供的3mL缓冲液中来制备颜色试剂。</li>
	<li>装入 10 x 10 x 45 mm 的聚苯乙烯比色皿，加入 3 mL 彩色试剂。</li>
	<li>使用纯色试剂作为空白条件（空白）。加入20μL LUV样品（测试）或20μL已知胆碱浓度的标准溶液（标准）。</li>
	<li>充分混合并在所有条件（空白，测试和标准）下在37°C下孵育5分钟。</li>
	<li>用分光光度计在600nm处以空白溶液作为对照，测量测试样品和标准溶液的吸光度（光密度，OD）。</li>
	<li>检查OD值，以便能够估计与已知浓度的标准相比，胆碱当量的LUV，C[LUV]的脂质浓度。</li>
	<li>使用以下等式执行计算: C[LUV] （摩尔 / 升） = （OD 样品 / OD 标准） x C[标准] （摩尔 / 升） 注意:磷脂定量试剂盒提供了氯化胆碱（139.6 mg / l）标准溶液，浓度为54 mg / dL，对应于C[标准]的摩尔浓度=3.87 mmol/ L. OD样品和OD标准品分别是在600nm处测量的LUV和胆碱溶液的吸光度。</li>
</ol>4. LUV尺寸和均匀性的表征
<ol><li>使用DLS仪器进行测量，以确定LUV尺寸（以nm为单位）和多分散指数（PdI）。</li>
	<li>通过指示溶剂/缓冲液和所用比色皿的粘度来对适当的"标准操作程序"（SOP）进行编程。</li>
	<li>将500μLLUV溶液置于聚苯乙烯半微量比色皿中。</li>
	<li>将聚苯乙烯半微量比色皿插入DLS仪器中。</li>
	<li>在室温下，根据颗粒分布的平均尺寸（Z平均值）和均匀性（多分散指数，PdI）测量尺寸分布。</li>
	<li>所有结果都是通过三个重复周期进行的两次独立测量获得的。 注意:LUV 的标准值的平均大小为 137 ± 7 nm，PdI 为 0.149 ± 0.041。</li>
</ol>5. 制备肽溶液
<ol><li>准备肽的储备溶液，应对其进行泄漏测定分析。</li>
	<li>将肽粉（纯度>95%）溶解在纯水（例如，1mg肽在500μL纯水中）。 注意:建议在纯水中稀释肽并避免二甲基亚砜（DMSO）增溶，这可能会诱发伪影（例如，膜透化20）。</li>
	<li>涡旋肽溶液5 s。</li>
	<li>在水超声处理浴中超声处理肽溶液5分钟，然后在12，225×g下离心5分钟。 收集上清液进行浓度测定。</li>
	<li>测量三种独立肽稀释液在280nm处的吸光度，然后使用其摩尔消光系数ε（取决于肽序列中的色氨酸和酪氨酸含量）和Beer-Lambert规则计算肽浓度。 注意:如果肽含有色氨酸和酪氨酸，则根据色氨酸ε = 5，690 M -1 cm-1和酪氨酸ε = 1，280 M-1cm-1计算摩尔消光系数ε。如果肽序列不含色氨酸或酪氨酸，则可以进行其他比色测定来测量浓度（例如，BCA或Bradford）。</li>
	<li>将肽溶液在纯水中稀释至100μM的最终溶液并储存在4°C。 注意:在纯水中，在4°C储存期间不会发生肽降解。然而，肽浓度应每2周测量一次，以确保不会发生水分蒸发。</li>
</ol>6. 荧光泄漏测定
<ol><li>荧光泄漏测定在室温下在光谱仪上测量。激发和发射波长分别固定在Ex = 360 nm±3 nm和Em = 530 nm±5 nm。</li>
	<li>在石英荧光比色皿中将1 mL HEPES缓冲液2中的LUV稀释至终浓度为100μM。在实验过程中加入磁力搅拌器使溶液均质化。</li>
	<li>在前100秒内，在t = 0 s和t = 99 s之间单独测量LUV，以便访问背景荧光。 注意:在整个实验（15分钟）期间，也可以单独测量LUV，以便访问背景荧光和潜在的泄漏。</li>
	<li>此后，在接下来的900秒（15分钟）中，在加入肽溶液等分试样时，测量泄漏作为荧光强度的增加。该方案对测试的肽浓度从0.1μM到2.5μM进行。</li>
	<li>最后，通过加入1μLTriton X-100（0.1%，v / v）来溶解LUV，从而实现100%荧光，从而在t = 1，000 s和t = 1，100 s之间完全未淬火的探针。</li>
</ol>7. 泄漏量化
<ol><li>抑制 t = 1，090 s 后获得的值，以便为每个测试条件保持相同的点数。</li>
	<li>通过使t = 0 s和t = 49 s（仅LUV）之间的50个点的平均值来计算最小荧光Fmin。</li>
	<li>计算最大荧光Fmax，方法是在t = 1，041 s和t = 1，090 s之间取50个点的平均值（使用Triton X-100的LUV）。</li>
	<li>根据以下等式计算每个时间点（t = x）的泄漏百分比（%泄漏）: 泄漏百分比（t=x） = （F（t=x） - F最小值 ） / （F最大 值 - F最小值） x 100</li>
	<li>计算相同条件下使用不同 LUV 制备（n ≥ 2）获得的值的平均值和标准偏差。</li>
	<li>绘制泄漏百分比 %Leak（t=x），以时间 （s） 的函数表示。</li>
</ol>

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作者没有利益冲突。

所提出的荧光泄漏测定是一种简单快速的方法，用于解决细胞穿透肽的膜不稳定。它很容易做到，还可以间接比较不同的膜相互作用肽或其他膜相互作用分子。关于方案的关键步骤，由于该测定提供了基线（仅LUV）和最大荧光释放（Triton条件）之间的相对值，我们通常使用磷脂定量试剂盒评估LUV的浓度，该试剂盒仅估计LUV的胆碱贡献。然而，也可以包括更准确地测量LUV浓度，方法是通过Rouser及其同事27或Bartlett28所描述的酸消解来确定总磷含量，或者通过使用硫氰酸亚铁铵络合磷脂29的比色法。在我们手中，它对泄漏的相对定量和膜相互作用肽之间的比较没有影响。
关于LUV的使用，人们还应该注意到控制LUV基线状态的重要性，以便检查它们是否可用以及它们是否已经显示泄漏（其特征在于荧光的连续增加）。同样，重要的是测量每个肽孵育后的最大荧光释放，以确保不会由于不希望的淬灭过程（例如，肽/染料猝灭）而记录假阴性结果。
一般来说，根据DLS表征，LUV在1周内非常稳定，不应该显示太高的荧光背景。在这种情况下，通过凝胶过滤纯化ANTS / DPX负载的脂质体也是一个关键点，因为能够形成LUV，而不会产生可能导致背景或产生荧光高估的残留荧光。此外，强烈建议通过包括始终可以提供相同泄漏百分比的特定阳性对照来校准方案。这通过不同的测量构成了内部控制，这可能会加强每次测试的LUV表征并增加统计数据。
尽管荧光泄漏测定法可以快速比较CPP的膜不稳定，但它在肽 - 膜相互作用的解释方面受到限制，因为某些肽能够与脂质双分子层相互作用而不会引起任何膜干扰或泄漏。对于这些肽，强烈建议使用具有特定荧光标记物的额外实验，以实现FRET30。
还应该注意的是，可以使用其他荧光染料/淬灭剂对（例如，Tb3 +/ DPA31）。这两种方法都有其不便之处:铽（III）在水中不是很溶，不能在磷酸盐存在下使用，而ANTS淬火不是DPX浓度的线性函数。此外，其他自淬灭材料，如70mM钙黄绿素溶液32 或葡聚糖-PTS33， 可以根据分析的肽或化合物引起的膜缺陷进行处理。
最后，这种荧光泄漏测定的主要优点是能够测试多种潜在的膜相互作用分子以及不同的膜组成，例如内体膜模拟物[例如，二油酰基磷脂酰胆碱（DOPC）/二油酰基磷脂酰乙醇胺（DOPE）/大豆（PI）/双（单油基甘油）磷酸酯（LBPA）的磷脂酰肌醇，摩尔比为5:2:1:2]34，线粒体膜模拟[例如，46.5%DOPC，28.5%磷脂酰乙醇胺（PE），9%磷脂酰肌醇（PI），9%磷脂酰丝氨酸（PS）和7%心磷脂（CL）35 或任何其他所需的脂质双层组合物。然而，首先必须确保在实验和储存期间具有接近100nm的恒定平均尺寸的囊泡的稳定性（没有LUV融合，聚集或沉淀，没有脂质从悬浮液中脱落，没有负膜曲率等）。
此外，与提取的膜（红细胞，线粒体等）相比，该方法的优点是在没有蛋白质的情况下使用纯化的表征良好的脂质。脂质双层组成（血浆、内体或线粒体膜）的控制也使特定膜蛋白（质子泵）的插入成为可能。此外，控制LUV内部和外部环境的能力允许对膜干扰/泄漏进行清晰的解释。与黑脂膜实验36相比，其还可以可视化膜透化事件，这种泄漏方案提供了更简单的膜活性肽筛选方法。
总之，这种简单的方法有利于快速鉴定导致膜不稳定的强肽/膜相互作用。它可以用于研究CPP的内化机制，以及更一般的"膜活性肽"方法，如致纤维肽或抗菌肽。
一般来说，CPP用于到达细胞质的主要途径的表征非常复杂，需要几种不同的方法，从生物物理学到细胞生物学。例如，对WRAP内化的研究揭示了进入细胞的不同机制（内吞与直接易位）之间的平衡，并且与其他方法相关的荧光泄漏测定有助于支持直接渗透过程7。

在九十年代发现它们之后，细胞穿透肽（CPPs）被开发用于促进货物通过质膜的高效细胞递送1，2。CPP通常是短肽，通常为8至30个氨基酸，具有多种来源。它们首先被定义为"直接易位"携带者，这意味着它们能够穿过质膜并将货物转移到细胞中，而不受任何内吞途径的影响，既不要求能量也不参与受体。然而，进一步的研究表明，这些最初的观察结果主要来自由于实验伪影和/或使用甲醇3的固定方案而导致的荧光高估。如今，人们普遍认为CPP摄取是通过内吞作用和能量无关的易位4，5，6，7发生的，这取决于不同的参数，例如货物的性质，CPP和货物之间的使用链接，研究的细胞系等。
CPP可以根据两种策略用作转染剂，要么涉及CPP与其货物之间的化学链接（共价策略）或静电/疏水相互作用（非共价策略）8，9，10，11。尽管这两种策略都显示出它们在几种货物的细胞转移中的效率，但对CPP内化机制的理解仍然存在争议，并且内吞作用途径或直接渗透之间的平衡仍然难以测量12，13。虽然有一套实验工具和策略可以清楚地解决内吞过程的参与问题，但直接易位似乎更难表征，因为它意味着与质膜组分的更离散的相互作用。生物膜通常由许多组分组成，从磷脂到膜蛋白，这些组分可能根据细胞类型和/或环境（应激条件，细胞分裂等）而变化。这种组成的多样性，以及因此缺乏通用细胞膜模型，无法以单一方式进行研究。然而，为了规避这些限制，用人造膜或膜提取物开发了分步方法。从小型单层囊泡到单层方法，每个模型显然都与回答特定问题14，15相关。其中，大型单层囊泡（LUV）构成适当的膜模拟模型，以研究肽/膜相互作用作为内化过程的关键点。
在这种情况下，以下方案描述了通过监测阴离子荧光染料及其封装在脂质体中的相应多阳离子淬灭剂来研究肽和肽/膜相互作用对LUV完整性的影响。该工具用于研究CPP /膜相互作用，以了解它们是否能够进行直接膜易位。虽然通常用于比较不同的膜相互作用肽，但这种LUV荧光泄漏测定也可用于研究CPPs-cargo偶联物（共价策略）和CPP:cargo复合物（非共价策略）。
因此，本方案将首先以最近开发的色氨酸（W）-和精氨酸（R）富两性肽（WRAP）16为例。WRAP能够形成基于肽的纳米颗粒，以快速有效地在几个细胞系中递送小干扰RNA（siRNA）16。监测单独使用WRAP肽或siRNA负载的WRAP基纳米颗粒的荧光泄漏特性，以表征其细胞内化机制。结果表明，它们的内化机制主要涉及直接易位7。在第二个例子中，WRAP肽与蛋白质/蛋白质干扰肽iCAL36（WRAP-iCAL36）17共价偶联，并且在荧光泄漏测定中将其破坏膜稳定性的能力与iCAL36偶联到另一种CPP的Interrin 18（Penetratin-iCAL36）进行比较。
最后，将从技术角度和生物学相关性的角度讨论该方法的优点和局限性。

<table><tbody><tr><td>25 mL glass round-bottom flask</td><td>Pyrex</td><td></td><td></td></tr><tr><td>8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS)</td><td>Invitrogen</td><td>A350</td><td>Protect from light&amp;nbsp;</td></tr><tr><td>Chloroform&amp;nbsp;</td><td>Sigma-Aldrich</td><td>288306</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>DOPC (dioleoylphosphatidylcholine)</td><td>Avanti Polar</td><td>850375P</td><td>Protect from air</td></tr><tr><td>Extruder</td><td>Avanti Polar</td><td>610000</td><td></td></tr><tr><td>Fluorimeter</td><td>PTI Serlabo</td><td></td><td></td></tr><tr><td>50 &amp;micro;L glass syringe</td><td>Hamilton</td><td>705N</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>LabAssay Phospholipid&amp;nbsp;</td><td>WAKO&amp;nbsp;</td><td>296-63801</td><td></td></tr><tr><td>liquid chromatography column</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Methanol</td><td>Carlo Erba</td><td>414902</td><td></td></tr><tr><td>Nuclepore polycarbonate membrane (0.1 &amp;micro;m pore size, 25 mm diameter)</td><td>Whatman</td><td>800309</td><td></td></tr><tr><td>polystyrene cuvette, 10 x 10 x 45 mm</td><td>Grener Bio-One</td><td>614101</td><td></td></tr><tr><td>polystyrene semi-micro cuvette, DLS</td><td>Fisher Scientific</td><td>FB55924</td><td></td></tr><tr><td>p-xylene-bispyridinium bromide (DPX)</td><td>Invitrogen</td><td>X1525</td><td>Protect from light&amp;nbsp;</td></tr><tr><td>quartz fluorescence cuvette</td><td>Hellma</td><td>109.004F-QS</td><td></td></tr><tr><td>rotavapor system&amp;nbsp;</td><td>Heidolph</td><td>Z334898</td><td></td></tr><tr><td>Sephadex G-50 resin</td><td>Amersham</td><td>17-0042-01</td><td></td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium chlorid (NaCl)</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Sonicator bath USC300T</td><td>VWR</td><td>142-6001</td><td></td></tr><tr><td>Sphingomyelin</td><td>Avanti Polar</td><td>860062P</td><td>Protect from air</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Eromedex</td><td>2000-B</td><td></td></tr><tr><td>Zetaziser NanoZS&amp;nbsp;</td><td>Malvern</td><td>ZEN3500</td><td></td></tr></tbody></table>

fluorescent leakage assay to investigate membrane destabilization by cell-penetrating peptide

细胞穿透肽（CPP）被定义为能够穿过质膜并将货物转移到细胞中的载体。这种活性所需的主要共同特征之一是CPP与质膜（脂质）的相互作用，特别是与膜本身的细胞外基质（硫酸肝素）的相互作用。事实上，独立于直接易位或内吞作用依赖性内化，脂质双层在质膜水平和细胞内转量水平（内体囊泡）参与内化过程。在本文中，我们提出了一个详细的方案，描述了大型单层囊泡（LUV）配方，纯化，表征和在荧光泄漏测定中的应用的不同步骤，以检测可能的CPP膜不稳定/相互作用，并解决它们在内化机制中的作用。产生具有反映质膜含量的脂质组合物的LUV，以便封装荧光染料和淬灭剂。因此，在阴囊外培养基中添加肽和诱导LUV上的肽 - 膜相互作用可能以剂量依赖性方式诱导荧光的显着增加，从而揭示泄漏。这里提供了最近开发的色氨酸（W）和精氨酸（R）丰富的两性肽（WRAP）的例子，它们在各种细胞系中显示出快速有效的siRNA递送。最后，讨论了这些相互作用的性质和对脂质的亲和力，以了解和改善膜易位和/或内体逃逸。

荧光泄漏测定的原理如图 1所示。详细地，用感兴趣的生物分子处理封装荧光染料和淬灭剂（无荧光信号）的大型单层囊泡（LUV）。由于肽与脂质膜的相互作用，这可能意味着膜通透性，重组甚至破裂，荧光染料和淬灭剂从LUV中释放出来。缓冲液中的后续稀释导致荧光信号增加。
尽管该方案显示了对游离肽的测试，但该系统的优势在于能够测试货物偶联肽，基于肽的纳米颗粒或其他生物聚合物，这些生物聚合物被怀疑会破坏脂质膜的稳定性。虽然需要对方案进行初步优化，特别是在测试的分子方面，但这种荧光泄漏测定可以扩展到各种各样的膜相互作用组分。在本方案中，我们显示了使用CPP及其复杂（非共价策略）或共轭（共价策略）形式获得的一些结果。以下例子暗示了单独使用WRAP以及siRNA负载的WRAP基纳米颗粒和两种不同的肽偶联物（WRAP-iCAL3617 和Intelationin-iCAL36）。
关于非共价策略，在存在LUV的情况下，用肽及其相应的siRNA负载纳米颗粒进行荧光泄漏测定如图2所示。囊泡由反映科纳特等人21中描述的质膜的DOPC / SM / Chol（4:4:2）的混合物组成，通常用于直接评估游离WRAP和WRAP基纳米颗粒7的脂质双层相互作用和/或转导性质的可能性。在没有肽的情况下，没有观察到泄漏（前100秒的基线）。在LUV上添加游离WRAP可诱导荧光显着增加，从而显示重要的LUV泄漏和蚂蚁释放。15分钟后，在使用浓度（2.5μM）的WRAP肽下获得与Triton条件（阳性对照）相比的67.8%±0.4%的泄漏。这里应该注意的是，还测试了几种不同的浓度，并揭示了剂量依赖性荧光的增加，对应于剂量依赖性LUV泄漏7。相反，当WRAP以相同的浓度与siRNA组装以形成基于肽的纳米颗粒时，与游离肽相比，泄漏量弱1.5倍（40.5%±0.5%）（图2）。RICK肽（60%）或RICK:siRNA纳米颗粒（28%）的泄漏值相似。游离肽与纳米颗粒之间的值差异可以通过以下事实来解释:当参与纳米颗粒时，肽的很大一部分参与与siRNA的直接相互作用，从而降低了肽与脂质相互作用的可用性。
关于共价策略，在存在LUV的情况下使用CPP偶联物进行荧光泄漏测定如图3所示。使用相同的LUV组合物[DOPC / SM / Chol（4:4:2）]，应用两种偶联肽:WRAP-iCAL36和Inspirein-iCAL36。如前所述，在没有肽的情况下没有观察到泄漏。在注射2.5μM的Interin-iCAL36后15分钟，未检测到显着的荧光增加（2.3%±0.7%），而添加2.5μM的WRAP-iCAL36诱导净泄漏，其特征在于非常强的荧光信号（85.8%±11.1%）17。这些观察结果表明，对于某些肽或偶联物，可能不会发生荧光泄漏，表明没有肽/膜相互作用或没有脂质双层紊乱。这符合先前发表的结果表明，渗透蛋白和Tat都无法破坏膜23，24，25的稳定性。应该强调的是，CPP的膜不稳定特性可能会根据耦合货物26而变化。
此外，尽管WRAP-iCAL36引起强烈泄漏，但其他研究并未显示特定的细胞内化，表明这些偶联物仍保留在质膜17的脂质双层内。与WRAP纳米颗粒相反，我们假设WRAP-cargo偶联物能够通过粘附在脂质链之间或形成孔隙来破坏LUV膜的稳定性。
这些结果表明，荧光泄漏测定可能揭示某些CPP发展肽/膜相互作用的能力，这可能导致或多或少明显的膜通透性。此外，无论货物交付的策略（纳米颗粒 与 共轭物）都可能发生这些相互作用。相反，该方法不区分不诱导任何荧光泄漏的CPP是否仍可能与脂质的双层或生物膜相互作用。这种行为需要额外的方法，例如zeta电位测量，肽和膜之间的FRET，或色氨酸荧光实验，仅举几个例子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62028/62028fig01.jpg"> 图1:荧光泄漏测定的原理。 LUV装载了白色的荧光染料（ANTS）和灰色的相应淬灭剂（DPX）。在没有肽（红色）的情况下，没有观察到荧光信号，因为ANTS荧光被DPX淬灭。在LUV上添加肽诱导膜通透性以及随后的ANTS和DPX的释放导致ANTS荧光（黄色）的显着增加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62028/62028fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62028/62028fig02.jpg"> 图2:用游离WRAP和WRAP:siRNA纳米颗粒进行荧光泄漏测定。单独肽和siRNA负载的纳米颗粒以2.5μM的肽浓度应用于LUV上，WRAP基纳米颗粒以肽:siRNA摩尔比为20（R 20）配制，如Konate等人7，16所述。黑色箭头分别显示肽（纳米颗粒）和Triton X-100的注射。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62028/62028fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62028/62028fig03.jpg"> 图3:使用WRAP-iCAL36和渗透蛋白-iCAL36偶联物的荧光泄漏测定。 将偶联物以2.5μM的浓度施加在LUV上，黑色箭头分别显示肽和Triton X-100的注射。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62028/62028fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide at JoVE.com

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

荧光泄漏测定是一种简单的方法，可以研究肽/膜相互作用，以了解它们在几个生物过程中的参与，特别是细胞穿透肽在直接细胞易位过程中扰乱磷脂双层的能力。

荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

fluorescent-leakage-assay-to-investigate-membrane-destabilization

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.2K 次观看.  INSERM U1046 - CNRS UMR 9214 - University of Montpellier. 荧光泄漏测定是一种简单的方法，可以研究肽/膜相互作用，以了解它们在几个生物过程中的参与，特别是细胞穿透肽在直接细胞易位过程中扰乱磷脂双层的能力。

视频: Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

构建环状细胞穿透肽以增强生物屏障的穿透力

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

科研

化学

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

双光子激光显微镜在细胞膜修复中的应用

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

免疫与感染

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

生物学

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins

Source: Guo, L. et. al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54266">Assays for the Degradation of Misfolded Proteins in Cells.</a> J. Vis. Exp. (2016)
This video describes an in vitro fluorescence-based microplate assay to study the degradation kinetics of a fluorescently-labeled misfolded luciferase mutant protein expressed in the nuclei of transfected mammalian cells. The assay involves the treatment of cells expressing the fluorescent mutant protein with a translation inhibitor and a proteasome inhibitor to assess the proteasome-mediated degradation of the misfolded protein.

基于荧光微孔板的放线菌酮追踪测定:一种监测荧光核错误折叠蛋白降解动力学的技术

Source: Guo, L. et. al., Assays for the Degradation of Misfolded Proteins in Cells. J. Vis. Exp. (2016)
This video describes an in vitro ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

一种用于筛选蛋白酶蛋白水解活性的荧光肽切割测定法

1. Designing and Preparing the Peptides

	Acquire the sequence of the fusion protein of interest from a public database such as NCBI ...

Immunology

Immunodiagnostics

Specialized Assays

Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane

Source: Konate, K. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62028">Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide.</a> J. Vis. Exp. (2020)
This video discusses the fluorescence leakage assay to study the interactions of cell-penetrating peptides with cell membranes using cell membrane mimics &#8212; large unilamellar vesicles, or LUVs, encapsulated with a fluorescent dye and a quencher.

荧光泄漏测定研究细胞穿透肽与膜的相互作用

Source: Konate, K. et al. Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide. J. Vis. Exp. (2020)
This video ...