细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究
Summary
该协议描述了模拟单脂质和多脂质囊泡，支持脂质双分子层和悬浮脂质双分子层的细胞的形成。这些 体外 模型可以适应于结合各种脂质类型，并可用于研究各种分子和大分子相互作用。
Abstract
模型细胞膜是一种有用的筛选工具，其应用范围从早期药物发现到毒性研究。细胞膜是所有细胞类型的重要保护屏障，将内部细胞成分与细胞外环境分开。这些膜主要由脂质双层组成，其包含外部亲水性头基团和内疏水尾基团，以及各种蛋白质和胆固醇。脂质本身的组成和结构在调节生物功能方面起着至关重要的作用，包括细胞与细胞微环境之间的相互作用，其中可能含有药物，生物毒素和环境毒物。在这项研究中，描述了配制单脂质和多脂支持和悬浮细胞模仿脂质双分子层的方法。以前，开发了单脂磷脂酰胆碱（PC）脂质双分子层以及多脂胎盘滋养层启发的脂质双分子层，用于理解分子相互作用。在这里，将介绍实现这两种类型的双层模型的方法。对于模拟多脂双分子层的细胞，首先通过从原代细胞或细胞系中提取脂质，然后通过液相色谱-质谱（LC-MS）确定所需的脂质组成。使用该组合物，使用薄膜水合和挤出方法制备脂质囊泡，并表征其流体动力学直径和zeta电位。然后，可以使用具有耗散监测功能的石英晶体微量天平（QCM-D）和多孔膜分别用于平行人工膜渗透率测定（PAMPA）形成支撑和悬浮的脂质双分子层。代表性结果突出了 体外 细胞膜脂质双层模型的再现性和多功能性。所提出的方法可以帮助快速，方便地评估各种分子和大分子与细胞膜的相互作用机制，例如渗透，吸附和嵌入，有助于筛选候选药物和预测潜在的细胞毒性。
Introduction
细胞膜主要由磷脂，胆固醇和蛋白质组成，是所有活细胞1的重要组成部分。在脂质两亲性的驱动下，细胞膜起到保护屏障的作用，并调节细胞如何与其周围环境相互作用2。一些细胞过程依赖于膜1、2的脂质和蛋白质组成。例如，细胞膜相互作用对于有效的药物递送很重要3。药品、生物制剂、纳米材料、生物毒素和环境毒物都会影响细胞膜的完整性，从而影响细胞功能4。基于细胞膜脂质组成的体外细胞模拟膜模型的构建有可能提供简单的工具，以大大增强对这些材料对细胞的潜在影响的研究。
模型脂质双层包括脂质囊泡、支持脂质双层和悬浮脂质双层。负载脂质双分子层是磷脂细胞膜的模型，通常用于生物技术应用，其中脂质囊泡在支撑的底物材料5，6，7，8，9上破裂。用于监测双层形成的一种常用技术是具有耗散监测功能的石英晶体微量天平（QCM-D），它检查囊泡的吸附与原位8，10，11，12，13，14的体积液体性质的比较.以前，QCM-D已被用于证明在流动条件下，一旦在表面上实现磷脂酰胆碱（PC）脂质囊泡的关键囊泡覆盖，它们就会自发破裂成刚性脂质双层15。先前的工作还研究了具有不同脂质组成的支持脂质双层形成16，脂质蛋白17，18，19的掺入，并利用聚合物垫20，产生能够模仿细胞膜功能各个方面的负载脂质双层。
脂质双分子层已被用于通过改变磷脂，胆固醇和糖脂成分来模仿从亚细胞到器官水平（包括线粒体，红细胞和肝细胞膜）的各种生物屏障21。这些更复杂的多脂质囊泡可能需要额外的方法来实现囊泡破裂，这取决于脂质组成。例如，以前的研究已经利用来自丙型肝炎病毒的非结构蛋白5A的α螺旋（AH）肽，通过破坏吸附的脂质囊泡22，23来诱导双层的形成。使用这种AH肽，支持脂质双层模仿胎盘细胞先前已经形成24。支持脂质双分子层在生物医学应用中的巨大潜力已经通过分子和纳米颗粒运输25、26、环境毒物相互作用27、蛋白质组装和功能17、18、19、肽排列和插入28、29、药物筛选30和微流体平台31的研究得到了证明。
悬浮脂质双分子层已通过平行人工膜渗透率测定法（PAMPA）用于药物筛选研究，其中脂质双分子层悬浮在多孔疏水插入物32，33，34，35上。PAMPA脂质模型已经开发用于不同的生物界面，包括血脑，颊，肠和透皮界面36。通过结合支持的脂质双分子层和PAMPA技术，可以彻底研究化合物在所需组织或细胞类型的脂质成分中的吸附，渗透性和包埋。
该协议描述了 体外 细胞膜脂质双层模型的制备和应用，以研究几种分子相互作用。详细介绍了单脂和多脂支持和悬浮脂质双分子层的制备。为了形成支持的脂质双层，首先使用薄膜水合和挤出方法开发脂质囊泡，然后进行物理化学表征。讨论了使用QCM-D监测形成支持的脂质双层和制造用于PAPA的悬浮脂质膜。最后，检查用于开发更复杂的细胞模拟膜的多脂囊泡。使用两种类型的制造脂质膜，该协议演示了如何使用该工具来研究分子相互作用。总体而言，该技术构建了具有高可重复性和多功能性的模拟脂质双分子层的细胞。
Protocol
1. 发展单脂囊泡 薄膜水合法 脂质储备溶液的制备和储存注：使用氯仿的所有步骤都需要在化学通风橱中执行。氯仿应始终使用溶剂安全的碳纤维移液器吸头进行移液。含有氯仿的溶液应始终储存在玻璃瓶中。 通过将适当体积的氯仿加入含有脂质粉末的小瓶中并充分混合，制备10mg / mL脂质储备溶液。例如，将20毫升氯仿加入200毫克L-α磷脂酰胆碱（鸡蛋，鸡肉）（鸡蛋PC）。?…
Representative Results
该协议详细介绍了形成支持和悬浮脂质双层的方法（图1）。形成支持脂质双层的第一步是发育脂质囊泡。小型挤出机允许制备小体积的脂质囊泡（1 mL或更少），而大型挤出机允许在一批中制备5-50 mL脂质囊泡。由小型或大型挤出机形成的单脂质囊泡的尺寸分布如图 2A所示。由于大型挤出机使用高压N2 气体将囊泡溶液推过聚碳酸酯膜，脂质囊泡?…
Discussion
该协议允许形成脂质囊泡，支持的脂质双层和悬浮脂质双层。在这里，提出了形成这些结构的关键步骤。当形成脂质囊泡时，重要的是挤出高于脂质39的转变温度 。当低于转变温度时，脂质物理上存在于其有序的凝胶相39中。在这个有序阶段，碳氢化合物脂质尾部完全伸展，允许紧密包装，使挤出具有挑战性39。当加热到转变温度以上时，脂质…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本材料基于美国国家科学基金会根据授予A.S.的第1942418号赠款以及授予C.M.B.H.的国家科学基金会研究生研究奖学金（第1644760号赠款）支持的工作。本材料中表达的任何意见，发现和结论或建议均为作者的观点，并不一定反映美国国家科学基金会的观点。作者感谢Noel Vera-González博士的脂质囊泡表征数据采集。作者感谢罗伯特·赫特教授（布朗大学）使用他的Zetasizer。作者感谢布朗大学质谱设施，特别是沈敦立博士在量化脂质组成方面的帮助。
Materials
| 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, 16:0-18:1 PC) | Avanti Polar Lipids | 850457 | |
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS, 16:0-18:1 PS) | Avanti Polar Lipids | 840034 | |
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE) | Avanti Polar Lipids | 850757 | |
| 1,2-dioleoyl-sn-glycero-2-phospho-L-serine (DOPS, 18:1 PS) | Avanti Polar Lipids | 840035 | |
| 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 18:1 (Δ9-Cis) PC) | Avanti Polar Lipids | 850375 | |
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 18:1 (Δ9-Cis) PE) | Avanti Polar Lipids | 850725 | |
| 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:0 EPC (Cl Salt)) | Avanti Polar Lipids | 890703 | |
| 3 mL Luer-Loc syringes | BD | 309657 | |
| 40 mL sample vial, amber with polytetrafluoroethylene (PTFE)/rubber liner | Duran Wheaton Kimble | W224605 | |
| Acetonitrile | Sigma-Aldrich | 271004 | |
| Alconox | Fisher Scientific | 50-821-781 | |
| Ammonium formate | Millipore Sigma | LSAC70221 | |
| C18, 3.5 um x 50 mm column, SunFire | Waters | 186002551 | |
| Chloroform | Millipore Sigma | LSAC288306 | |
| Cuvette UV Micro LCH 8.5 mm, 50 um, RPK | Sarstedt | 67.758.001 | |
| Di(2-ethylhexyl) phthalate (DEHP) | Millipore Sigma | 36735 | |
| Dimethyl sulfoxide (DMSO) | Millipore Sigma | LSAC472301 | |
| Ethanol | Pharmco | 111000200 | |
| Filter supports, 10 mm | Avanti Polar Lipids | 610014 | Size for mini extruder |
| Folded capillary zeta cell | Malvern Panalytical | DTS1070 | |
| Isopropanol | Sigma-Aldrich | 190764-4L | |
| Kimwipes | Kimberly Clark | 34256 | |
| L-α-phosphatidylinositol (soy) (Soy PI) | Avanti Polar Lipids | 840044 | |
| L-α-phosphitidylcholine (Egg, Chicken) | Avanti Polar Lipids | 840051 | |
| LiposoFast ® LF-50 | Avestin, Inc. | ||
| Methanol | Sigma-Aldrich | 179337 – 4L | |
| Mini-extruder set with holder/heating block | Avanti Polar Lipids | 610000 | |
| MultiScreen-IP Filter Plate, 0.45 µm, clear, sterile | Millipore Sigma | MAIPS4510 | for PAMPA studies |
| Nitrogen gas, ultrapure | TechAir | NI T5.0 | |
| Nuclepore hydrophilic membranes, polycarbonate, 19 mm, 0.1 um | Whatman | 800309 | Size for mini extruder |
| Nuclepore hydrophilic membranes, polycarbonate, 25 mm, 0.1 um | Whatman | 110605 | Size for large extruder |
| Parafilm | Bemis | PM999 | |
| Phosphate buffer saline (PBS), 10x | Genesee Scienfitic | 25-507X | Dilute to 1x |
| Qsoft 401 software | Biolin Scientific | ||
| Quartz Crystal Microbalance with Dissipation Q-Sense Analyzer | Biolin Scientific | ||
| Scintillation vials, borosilicate glass vials, 20 mL | Duran Wheaton Kimble | 986561 | |
| Silicon Dioxide, thin QSensors | Biolin Scientific | QSX 303 | |
| Sodium chloride (NaCl) | Millipore Sigma | LSACS5886 | |
| Sodium dodecyl sulfate (SDS) | Fisher Scientific | BP166-100 | |
| Solvent Safe pipette tips | Sigma-Aldrich | S8064 | |
| Sphingomyelin (Egg, Chicken) | Avanti Polar Lipids | 860061 | |
| Trizma base | Millipore Sigma | LSACT1503 | |
| Trypsin-ethylenediaminetretaacetic acid | Caisson Labs | TRL01-6X100ML | |
| Whatman drain disc, 25 mm | Whatman | 230600 | Size for large extruder |
| Zetasizer ZS90 | Malvern Panalytical | ||
| Zetasizer 7.01 software | Malvern Panalytical |
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Citazione di questo articolo
Bailey-Hytholt, C. M., LaMastro, V., Shukla, A. Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions. J. Vis. Exp. (174), e62599, doi:10.3791/62599 (2021).