Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Summary

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Abstract

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

Introduction

Giant vesicles obtained from phospholipids (liposomes) have been widely used since the 1970s as the basic cell membrane model¹. In the late 1990s, vesicular morphologies obtained from the self-assembly of copolymers, named polymersomes in reference to their lipid analogs^2,3, rapidly appeared as an interesting alternative to liposomes that possess weak mechanical stability and poor modular chemical functionality. However, their cell biomimetic character is rather limited compared to liposomes since the latter are composed of phospholipids, the main component of the cell membrane. Further....

Protocol

1. Fabricating micropipettes

NOTE: Here, micropipettes with an inner diameter ranging from 6 to 12 µm and a taper length around 3-4 mm are necessary. A detailed method of manufacturing micropipette is described in the following.

1. Place the borosilicate glass capillary in the drawbar of the puller and fix one of the ends by tightening the knob.
2. Carefully slide the glass through the holes at the side of the heater chamber.
3. Tighten down the clamping knob at the other end.
4. Control the size of the tip and the taper length to achieve the desired specifications. For that, optimize technical parameters such ....

Results

With the protocol aforementioned, we have studied different synthetic giant unilamellar vesicle (GUV), obtained from a phospholipid: 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), a triblock copolymer: Poly(ethyleneoxide)-b-Poly(dimethylsiloxane)-b-Poly(ethyleneoxide) (PEO₁₂-b-PDMS₄₃-b-PEO₁₂) synthesized in a previous study¹³, and a diblock copolymer Poly(dimethylsiloxane)-b.......

Discussion

The coating of the micropipette is one of the key points to obtain reliable measurements. Adhesion of the vesicle to the micropipette must be prevented, and a coating is commonly used in literature^{17,18,19,20,21}, with BSA, β-casein or surfasil. Details of the coating procedure are rarely mentioned.

Dissolution of the BSA should.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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