荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 大型单层囊泡（LUV）的制备 准备LUV，用作荧光泄漏测定的细胞膜模拟物。 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?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 25 mL glass round-bottom flask | Pyrex | ||
| 8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS) | Invitrogen | A350 | Protect from light |
| Chloroform | Sigma-Aldrich | 288306 | |
| Cholesterol | Sigma-Aldrich | C8667 | |
| DOPC (dioleoylphosphatidylcholine) | Avanti Polar | 850375P | Protect from air |
| Extruder | Avanti Polar | 610000 | |
| Fluorimeter | PTI Serlabo | ||
| 50 µL glass syringe | Hamilton | 705N | |
| HEPES | Sigma-Aldrich | H3375 | |
| LabAssay Phospholipid | WAKO | 296-63801 | |
| liquid chromatography column | Sigma-Aldrich | ||
| Methanol | Carlo Erba | 414902 | |
| Nuclepore polycarbonate membrane (0.1 µm pore size, 25 mm diameter) | Whatman | 800309 | |
| polystyrene cuvette, 10 x 10 x 45 mm | Grener Bio-One | 614101 | |
| polystyrene semi-micro cuvette, DLS | Fisher Scientific | FB55924 | |
| p-xylene-bispyridinium bromide (DPX) | Invitrogen | X1525 | Protect from light |
| quartz fluorescence cuvette | Hellma | 109.004F-QS | |
| rotavapor system | Heidolph | Z334898 | |
| Sephadex G-50 resin | Amersham | 17-0042-01 | |
| Sodium azide (NaN3) | Sigma-Aldrich | S2002 | |
| Sodium chlorid (NaCl) | Sigma-Aldrich | S5886 | |
| Sonicator bath USC300T | VWR | 142-6001 | |
| Sphingomyelin | Avanti Polar | 860062P | Protect from air |
| Triton X-100 | Eromedex | 2000-B | |
| Zetaziser NanoZS | Malvern | ZEN3500 |
References
- Langel, U. . Handbook of Cell-Penetrating Peptides. , (2006).
- Deshayes, S., Morris, M. C., Divita, G., Heitz, F. Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cellular and Molecular Life Sciences CMLS. 62 (16), 1839-1849 (2005).
- Richard, J., et al. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. The Journal of Biological Chemistry. , (2003).
- Jones, A., Sayers, E. Cell entry of cell penetrating peptides: tales of tails wagging dogs. Journal of Controlled Release Official Journal of the Controlled Release Society. , (2012).
- Tünnemann, G., et al. Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. Journal of Peptide Science. 14 (4), 469-476 (2008).
- Jiao, C. -. Y., et al. Translocation and endocytosis for cell-penetrating peptide internalization. Journal of Biological Chemistry. 284 (49), 33957-33965 (2009).
- Deshayes, S., et al. Deciphering the internalization mechanism of WRAP:siRNA nanoparticles. Biochimica Et Biophysica Acta. Biomembranes. 1862 (6), 183252 (2020).
- Konate, K., et al. Optimisation of vectorisation property: A comparative study for a secondary amphipathic peptide. International Journal of Pharmaceutics. 509 (1-2), 71-84 (2016).
- Lehto, T., et al. Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells. Nucleic Acids Research. 42 (5), 3207-3217 (2014).
- Hoyer, J., Neundorf, I. Peptide vectors for the nonviral delivery of nucleic acids. Accounts of Chemical Research. 45 (7), 1048-1056 (2012).
- Milletti, F. Cell-penetrating peptides: classes, origin, and current landscape. Drug Discovery Today. 17 (15-16), 850-860 (2012).
- Mueller, J., Kretzschmar, I., Volkmer, R., Boisguerin, P. Comparison of cellular uptake using 22 CPPs in 4 different cell lines. Bioconjugate Chemistry. 19 (12), 2363-2374 (2008).
- Ramaker, K., Henkel, M., Krause, T., Röckendorf, N., Frey, A. Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs. Drug Delivery. 25 (1), 928-937 (2018).
- Maget-Dana, R. The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes. Biochimica Et Biophysica Acta. 1462 (1-2), 109-140 (1999).
- Alves, A. C., Ribeiro, D., Nunes, C., Reis, S. Biophysics in cancer: The relevance of drug-membrane interaction studies. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1858 (9), 2231-2244 (2016).
- Konate, K., et al. Peptide-based nanoparticles to rapidly and efficiently “Wrap ‘n Roll” siRNA into cells. Bioconjugate Chemistry. 30 (3), 592-603 (2019).
- Seisel, Q., Pelletier, F., Deshayes, S., Boisguerin, P. How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides. Biochimica Et Biophysica Acta. Biomembranes. 1861 (9), 1533-1545 (2019).
- Derossi, D., Joliot, A. H., Chassaing, G., Prochiantz, A. The third helix of the Antennapedia homeodomain translocates through biological membranes. Journal of Biological Chemistry. 269 (14), 10444-10450 (1994).
- Takayama, M., Itoh, S., Nagasaki, T., Tanimizu, I. A new enzymatic method for determination of serum choline-containing phospholipids. Clinica Chimica Acta; International Journal of Clinical Chemistry. 79 (1), 93-98 (1977).
- Notman, R., Noro, M., O’Malley, B., Anwar, J. Molecular basis for Dimethylsulfoxide (DMSO) action on lipid membranes. Journal of the American Chemical Society. 128 (43), 13982-13983 (2006).
- Konate, K., et al. Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery. 생화학. 49 (16), 3393-3402 (2010).
- Vaissière, A., et al. A retro-inverso cell-penetrating peptide for siRNA delivery. Journal of Nanobiotechnology. 15 (1), 34 (2017).
- Eiríksdóttir, E., Konate, K., Langel, &. #. 2. 2. 0. ;., Divita, G., Deshayes, S. Secondary structure of cell-penetrating peptides controls membrane interaction and insertion. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1798 (6), 1119-1128 (2010).
- Ziegler, A., Li Blatter, X., Seelig, A., Seelig, J. Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis. 생화학. 42 (30), 9185-9194 (2003).
- Thorén, P. E. G., Persson, D., Karlsson, M., Nordén, B. The Antennapedia peptide penetratin translocates across lipid bilayers – the first direct observation. FEBS Letters. 482 (3), 265-268 (2000).
- Mishra, A., et al. Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions. Proceedings of the National Academy of Sciences. 108 (41), 16883-16888 (2011).
- Rouser, G., Fleischer, S., Yamamoto, A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 5 (5), 494-496 (1970).
- Bartlett, G. R. Phosphorus assay in column chromatography. Journal of Biological Chemistry. 234 (3), 466-468 (1959).
- Stewart, J. C. M. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Analytical Biochemistry. 104 (1), 10-14 (1980).
- Spinella, S. A., Nelson, R. B., Elmore, D. E. Measuring peptide translocation into large unilamellar vesicles. Journal of Visualized Experiments: JoVE. (59), e3571 (2012).
- Wimley, W. C. Determining the effects of membrane-interacting peptides on membrane integrity. Cell-Penetrating Peptides. 1324, 89-106 (2015).
- Bárány-Wallje, E., Gaur, J., Lundberg, P., Langel, U., Gräslund, A. Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo. FEBS Letters. 581 (13), 2389-2393 (2007).
- van Rooijen, B. D., Claessens, M. M. A. E., Subramaniam, V. Membrane permeabilization by oligomeric α-Synuclein: in search of the mechanism. PloS One. 5 (12), 14292 (2010).
- Hassane, F. S., et al. Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides. Journal of Controlled Release: Official Journal of the Controlled Release Society. 153 (2), 163-172 (2011).
- Asciolla, J. J., Renault, T. T., Chipuk, J. E. Examining BCL-2 family function with large unilamellar vesicles. Journal of Visualized Experiments: JoVE. (68), e4291 (2012).
- Grewer, C., Gameiro, A., Mager, T., Fendler, K. Electrophysiological characterization of membrane transport proteins. Annual Review of Biophysics. 42 (1), 95-120 (2013).
Cite This Article
Konate, K., Seisel, Q., Vivès, E., Boisguérin, P., Deshayes, S. Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide. J. Vis. Exp. (166), e62028, doi:10.3791/62028 (2020).