通过电形成和微移液器测量其机械性能，对巨型Unilamellar混合囊泡的吸收
Summary
该协议的目标是通过微移液吸气可靠地测量巨型囊泡的膜机械性能。
Abstract
从磷脂和共聚物中获得的巨型囊泡可以在不同的应用中被利用：受控和靶向药物输送、用于诊断的生物传感器内的生物分子识别、人造细胞的功能膜以及生物启发微/纳米反应器的开发。在所有这些应用中，其膜特性的表征至关重要。在现有的表征技术中，由 E. Evans 开创的微移液器吸气允许测量膜的机械特性，如面积压缩模量、弯曲模量和解压应力和应变。在这里，我们介绍了从脂质或共聚物（或两者）的薄膜中获取巨型囊泡的所有方法和详细程序，微移液器的制造和表面处理，以及导致测量上述所有参数的吸气程序。
Introduction
从磷脂（脂质体）获得的巨型囊泡自20世纪70年代以来被广泛用作基本细胞膜模型1。在20世纪90年代后期，从共聚物的自组装中获得的晶型形态，根据其脂质类比2，3，被命名为聚合物体，迅速成为具有弱机械稳定性和模块化化学功能的脂质体的有趣替代品。然而，与脂质体相比，它们的细胞生物体性质相当有限，因为脂质体由磷脂组成，磷脂是细胞膜的主要成分。此外，在药物输送等应用中，其低膜渗透性可能是一个问题，需要控制物种通过膜的扩散。最近，磷脂与块共聚物的关联，设计混合聚合物脂质囊泡和膜已成为越来越多的研究课题4，5。其主要思想是设计实体，将每种成分（脂质双层的生物功能和渗透性）与聚合物膜的机械稳定性和化学多功能性协同结合，这些优势可在不同的应用中加以利用：受控和靶向药物输送、用于诊断的生物传感器内的生物分子识别、人造细胞的功能膜、生物启发微/纳米反应器的开发。
如今，不同的科学界（生物化学家、化学家、生物物理学家、物理化学家、生物学家）对开发更先进的细胞膜模型越来越感兴趣。在这里，我们的目标是尽可能详细地提出现有方法（电镀、微移液吸入）来获得和表征巨型囊泡的机械特性，以及最近”先进”的细胞膜模型，即混合聚合物脂质巨型囊泡4、5。
这些方法的目的是获得可靠的测量面积的可压缩性和弯曲膜膜，以及其解压应力和应变。测量巨型囊泡弯曲刚度的最常见的技术之一是基于直接视频显微镜观察的波动分析6，7;但这需要较大的可见膜波动，并且不会在厚膜（如聚合物体）上系统地获得。区域可压缩模量可以用兰缪尔·布洛杰特技术进行实验确定，但最常见的是单层8。微移液器吸气技术允许在一个实验中测量形成巨型单体囊泡 （GUV） 的双层体上的两种莫杜利。
以下方法适用于所有两栖分子或大分子，能够形成双层，因此，通过电形成体囊。这需要在电形成温度下的双层结构具有流体特性。
Protocol
1. 制造微移液器 注：此处，内径为 6 至 12 μm 且直径长约 3-4 mm 的微移液器是必需的。下面介绍了制造微移液的详细方法。 将硼硅酸盐玻璃毛细管放在拉拔器的拉杆中，通过拧紧旋钮来固定其中一个端部。 小心地将玻璃滑过加热器室侧面的孔。 拧紧另一端的夹紧旋钮。 控制尖端的大小和片长，以达到所需的规格。为此，优化加热温度、拉力、?…
Representative Results
根据上述协议， 我们研究了不同的合成巨型单片杆菌囊泡（GUV），从磷脂获得：2-oleoyl-1-palmitoyl-sn-甘油-3磷胆碱（POPC），三聚物共聚物：聚（环氧）-b-聚宝（二甲基硅氧烷）-b-聚氨酯（环氧乙烷）（PEO12-b-PDMS43-b-PEO12）在先前的研究13中合成，以及二聚基共聚Poly（二甲基硅氧烷）-b-Poly（二甲基硅?…
Discussion
微移液器的涂层是获得可靠测量的关键点之一。必须防止囊泡粘附在微移液器上，在文献中通常使用涂层17、18、19、20、21与BSA、β-酪蛋白或西沙西尔。很少提及涂装程序的详细信息。
在搅拌下，BSA 的溶解应至少进行 4 小时，以实现良好的溶解。然而，?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢ANR的财政支持（ANR Sysa）。
Materials
| Required equipment and materials for micropipette design | |||
| Borosilicate Glass Capillaries | World Precision Instruments | 1B100-4 | external and internal diameter of 1mm and 0.58 mm respectively. |
| Filament installed | Sutter Instrument Co. | FB255B | 2.5mm*2.5mm Box Filament |
| Flaming/Brown Micropipette Puller | Sutter Instrument Co. | Model P-97 | |
| Microforge | NARISHGE Co. | MF-900 | fitted with two objectives (10x and 32x) |
| Materials for coating pipette tips with BSA | |||
| Bovine Serum Albumin Fraction V (BSA) | Sigma-Aldrich | 10735078001 | |
| Disposable 1 ml syringe Luer Tip | Codan | 62.1612 | |
| Disposable 10 ml syringe Luer Tip | Codan | 626616 | |
| Disposable 5 ml syringe Luer Tip | Codan | 62.5607 | |
| Disposable acetate cellulose filter | Cluzeau Info Labo | L5003SPA | Pore size: 0.22µm, diameter: 25mm |
| Flexible Fused Silica Capillary Tubing | Polymicro Technologies. | TSP530660 | Inner Diameter 536µm, Outer Diameter 660µm, |
| Glucose | Sigma-Aldrich | G5767 | |
| Syringe 500 µL luer Lock GASTIGHT | Hamilton Syringe Company | 1750 | |
| Test tube rotatory mixer | Labinco | 28210109 | |
| Micromanipulation Set up | |||
| Aluminum Optical Rail, 1000 mm Length, M4 threads, X48 Series | Newport | ||
| Damped Optical Table | Newport | used as support of microscope to prevent external vibrations. | |
| Micromanipulator | Eppendorf | Patchman NP 2 | The module unit (motor unit for X, Y and Z movement) is mounted on the inverted microscope by the way of an adapter. |
| Micrometer | Mitutoyo Corporation | 350-354-10 | Digimatic LCD Micrometer Head 25,4 mm Range 0,001 mm |
| Plexiglass water reservoir (100 ml) | Home made | ||
| TCS SP5 inverted confocal microscope (DMI6000) equipped with a resonant scanner and a water immersion objective (HCX APO L 40x/0.80 WU-V-I). | Leica | ||
| X48 Rail Carrier 80 mm Length,with 1/4-20, 8-32 and 4-40 thread | Newport | ||
| Materials for sucrose and amphiphile solution preparation | |||
| 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine | Sigma-Aldrich | ||
| Chloroform | VWR | 22711.244 | |
| L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) | Sigma-Aldrich | 810146C | Rhodamine tagged lipid |
| Sucrose | Sigma-Aldrich | S7903 | |
| Electroformation set up | |||
| 10 µL glass capillary ringcaps | Hirschmann | 9600110 | |
| Disposable 1 ml syringe Luer Tip | Codan | 62.1612 | |
| H Grease | Apiezon | Apiezon H Grease | Silicon-free grease |
| Indium tin oxide coated glass slides | Sigma-Aldrich | 703184 | |
| Needle | Terumo | AN2138R1 | 0.8 x 38 mm |
| Ohmmeter (Multimeter) | Voltcraft | VC140 | |
| Toluene | VWR | 28676.297 | |
| Voltage generator | Keysight | 33210A |
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Citazione di questo articolo
Ibarboure, E., Fauquignon, M., Le Meins, J. Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration. J. Vis. Exp. (155), e60199, doi:10.3791/60199 (2020).