Name: obtention of giant unilamellar hybrid vesicles by electroformation and measurement of their mechanical properties by micropipette aspiration
Uploaded: 2020-01-19T13:00:00
Description: Watch this Scientific Journal Video about Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration at JoVE.com

The authors gratefully acknowledge the ANR for financial support (ANR Sysa).

1. Fabricating micropipettes
NOTE: Here, micropipettes with an inner diameter ranging from 6 to 12 &#181;m and a taper length around 3-4 mm are necessary. A detailed method of manufacturing micropipette is described in the following.
<ol>
	<li>Place the borosilicate glass capillary in the drawbar of the puller and fix one of the ends by tightening the knob.</li>
	<li>Carefully slide the glass through the holes at the side of the heater chamber.</li>
	<li>Tighten down the clamping knob at the...

<ol>
	<li>Bangham, A. D., Standish, M. M., Watkins, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+univalent+ions+across+the+lamellae+of+swollen+phospholipids.">Diffusion of univalent ions across the lamellae of swollen phospholipids.</a> Journal of Molecular Biology. 13 (1), (1965).</li><li>Discher, D. E., Eisenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+vesicles.">Polymer vesicles.</a> Science. 297 (5583), 967-973 (2002).</li><li>Hammer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes:+vesicles+from+block+copolymers.">Polymersomes: vesicles from block copolymers.</a> Annals of Biomedical Engineering. 28 (SUPPL. 1), (2000).</li><li>Le Meins, J. F., Schatz, C., Lecommandoux, S., Sandre, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+polymer/lipid+vesicles:+state+of+the+art+and+future+perspectives.">Hybrid polymer/lipid vesicles: state of the art and future perspectives.</a> Materials Today. 16 (10), 397-402 (2013).</li><li>Schulz, M., Binder, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+Hybrid+Lipid/Polymer+Vesicles+as+a+Novel+Membrane+Platform.">Mixed Hybrid Lipid/Polymer Vesicles as a Novel Membrane Platform.</a> Macromolecular Rapid Communications. 36, 2031-2041 (2015).</li><li>Schneider, M. B., Jenkins, J. T., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+fluctuations+of+large+quasi-spherical+bimolecular+phospholipid+vesicles.">Thermal fluctuations of large quasi-spherical bimolecular phospholipid vesicles.</a> Journal De Physique. 45 (9), 1457-1472 (1984).</li><li>Dimova, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+the+field+of+bending+rigidity+measurements+on+membranes.">Recent developments in the field of bending rigidity measurements on membranes.</a> Advances in Colloid and Interface Science. 208, 225-234 (2014).</li><li>Rodr&#237;guez-Garc&#237;a, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes:+smart+vesicles+of+tunable+rigidity+and+permeability.">Polymersomes: smart vesicles of tunable rigidity and permeability.</a> Soft Matter. 7 (4), 1532-1542 (2011).</li><li>Angelova, M. I., Dimitrov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+electroformation.">Liposome electroformation.</a> Faraday Discussions of the Chemical Society. 81, 303-311 (1986).</li><li>Dao, T. P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+properties+of+giant+polymer+and+lipid+vesicles+obtained+by+electroformation+and+pva+gel-assisted+hydration+methods.">Membrane properties of giant polymer and lipid vesicles obtained by electroformation and pva gel-assisted hydration methods.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 533, 347-353 (2017).</li><li>Pereno, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroformation+of+Giant+Unilamellar+Vesicles+on+Stainless+Steel+Electrodes.">Electroformation of Giant Unilamellar Vesicles on Stainless Steel Electrodes.</a> ACS omega. 2 (3), 994-1002 (2017).</li><li>Evans, E., Rawicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entropy-driven+tension+and+bending+elasticity+in+condensed-fluid+membranes.">Entropy-driven tension and bending elasticity in condensed-fluid membranes.</a> Physical Review Letters. 64 (17), 2094-2097 (1990).</li><li>Dao, T. P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+phase+separation+at+the+micron+scale+and+nanoscale+in+giant+polymer/lipid+hybrid+unilamellar+vesicles+(GHUVs).">Modulation of phase separation at the micron scale and nanoscale in giant polymer/lipid hybrid unilamellar vesicles (GHUVs).</a> Soft Matter. 13 (3), 627-637 (2017).</li><li>Helfrich, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+properties+of+lipid+bilayers:+theory+and+possible+experiments.">Elastic properties of lipid bilayers: theory and possible experiments.</a> Z Naturforsch C. 11 (11), 693-703 (1973).</li><li>Dao, T. P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+block+copolymers+and+phospholipids+to+form+giant+hybrid+unilamellar+vesicles+(GHUVs)+does+not+systematically+lead+to+&amp;#34;intermediate''+membrane+properties.">The combination of block copolymers and phospholipids to form giant hybrid unilamellar vesicles (GHUVs) does not systematically lead to &amp;#34;intermediate'' membrane properties.</a> Soft Matter. 14 (31), 6476-6484 (2018).</li><li>Shoemaker, S. D., Kyle Vanderlick, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+Studies+of+Lipid+Vesicles+in+the+L&#945;+and+L&#945;-Gel+Coexistence+Regimes.">Material Studies of Lipid Vesicles in the L&#945; and L&#945;-Gel Coexistence Regimes.</a> Biophysical Journal. 84 (2), 998-1009 (2003).</li><li>Longo, M. L., Ly, H. V., Dopico, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Membrane Lipids. , 421-437 (2007).</li><li>Chen, D., Santore, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+copolymer-phospholipid+vesicles:+phase+separation+resembling+mixed+phospholipid+lamellae,+but+with+mechanical+stability+and+control.">Hybrid copolymer-phospholipid vesicles: phase separation resembling mixed phospholipid lamellae, but with mechanical stability and control.</a> Soft Matter. 11 (13), 2617-2626 (2015).</li><li>Mabrouk, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+material+properties+of+giant+liquid+crystal+polymersomes.">Formation and material properties of giant liquid crystal polymersomes.</a> Soft Matter. 5, 1870-1878 (2009).</li><li>Henriksen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+behavior+of+membranes+with+sterols.">Universal behavior of membranes with sterols.</a> Biophysical Journal. 90 (5), 1639-1649 (2006).</li><li>Ly, H. V., Block, D. E., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Tension+Effect+of+Ethanol+on+Lipid+Bilayer+Rigidity,+Stability,+and+Area/Molecule:&#8201;+A+Micropipet+Aspiration+Approach.">Interfacial Tension Effect of Ethanol on Lipid Bilayer Rigidity, Stability, and Area/Molecule:&#8201; A Micropipet Aspiration Approach.</a> Langmuir. 18 (23), 8988-8995 (2002).</li><li>Bermudez, H., Hammer, D. A., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Bilayer+Thickness+on+Membrane+Bending+Rigidity.">Effect of Bilayer Thickness on Membrane Bending Rigidity.</a> Langmuir. 20, 540-543 (2004).</li></ol>

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 literature17,18,19,20,21, with BSA, &#946;-casein or surfasil. Details of the coating procedure are rarely mentioned.
Dissolution of the BSA should...

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 analogs2,3, 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. Furthermore, their low membrane permeability can be an issue in some applications like drug delivery where controlled diffusion of species through the membrane is required. Recently, the association of phospholipids with block copolymers to design hybrid polymer-lipid vesicles and membranes has been the subject of an increasing number of studies4,5. The main idea is to design entities that synergistically combine the benefits of each component (bio-functionality and permeability of lipid bilayers with the mechanical stability and chemical versatility of polymer membranes), which can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, development of bio-inspired micro-/nano-reactors.
Nowadays, different scientific communities (biochemists, chemists, biophysicists, physico-chemists, biologists) have increasing interest in development of a more advanced cell membrane model. Here, our goal is to present, as detailed as possible, existing methodologies (electroformation, micropipette aspiration) to obtain and characterize the mechanical properties of giant vesicles and the recent &#34;advanced&#34; cell membrane models that are hybrid polymer lipid giant vesicles4,5.
The purpose of these methods is to obtain reliable measurement of the area compressibility and bending moduli of the membrane as well as their lysis stress and strain. One of the most common techniques existing to measure bending rigidity of a giant vesicle is fluctuation analysis6,7, based on direct video microscope observation; but this requires large visible membrane fluctuation, and is not systematically obtained on thick membranes (e.g. polymersomes). Area compressibility modulus can be experimentally determined using the Langmuir Blodgett technique but most often on a monolayer8. The micropipette aspiration technique allows the measurement of both moduli on a bilayer forming giant unilamellar vesicle (GUV) in one experiment.
The following method is appropriate for all amphiphilic molecules or macromolecules able to form bilayers and, consequently, vesicles by electroformation. This requires a fluid character of the bilayer at the temperature of electroformation.

<table>
	<tbody>
		<tr>
			<td>Required equipment and materials for micropipette design</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td>external and internal diameter of 1mm and 0.58 mm respectively.</td>
		</tr>
		<tr>
			<td>Filament installed</td>
			<td>Sutter Instrument Co.</td>
			<td>FB255B</td>
			<td>2.5mm*2.5mm Box Filament</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>NARISHGE Co.</td>
			<td>MF-900</td>
			<td>fitted with two objectives (10x and 32x)</td>
		</tr>
		<tr>
			<td>Materials for coating pipette tips with BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>10735078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>62.1612</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 10 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>626616</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 5 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>62.5607</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable acetate cellulose filter</td>
			<td>Cluzeau Info Labo</td>
			<td>L5003SPA</td>
			<td>Pore size: 0.22&#181;m, diameter: 25mm</td>
		</tr>
		<tr>
			<td>Flexible Fused Silica Capillary Tubing</td>
			<td>Polymicro Technologies.</td>
			<td>TSP530660</td>
			<td>Inner Diameter 536&#181;m, Outer Diameter 660&#181;m,</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 500 &#181;L luer Lock GASTIGHT</td>
			<td>Hamilton Syringe Company</td>
			<td>1750</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube rotatory mixer</td>
			<td>Labinco</td>
			<td>28210109</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulation Set up</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Optical Rail, 1000 mm Length, M4 threads, X48 Series</td>
			<td>Newport</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Damped Optical Table</td>
			<td>Newport</td>
			<td></td>
			<td>used as support of microscope to prevent external vibrations.</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Eppendorf</td>
			<td>Patchman NP 2</td>
			<td>The module unit (motor unit for X, Y and Z movement) is mounted on the inverted microscope by the way of an adapter.</td>
		</tr>
		<tr>
			<td>Micrometer</td>
			<td>Mitutoyo Corporation</td>
			<td>350-354-10</td>
			<td>Digimatic LCD Micrometer Head 25,4 mm Range 0,001 mm</td>
		</tr>
		<tr>
			<td>Plexiglass water reservoir (100 ml)</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>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).</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X48 Rail Carrier 80 mm Length,with 1/4-20, 8-32 and 4-40 thread</td>
			<td>Newport</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for sucrose and amphiphile solution preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.sigmaaldrich.com/catalog/substance/2oleoyl1palmitoylsnglycero3phosphocholine760082685331611" target="_blank">2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine</a></td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>22711.244</td>
			<td></td>
		</tr>
		<tr>
			<td>L-&#945;-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl)</td>
			<td>Sigma-Aldrich</td>
			<td>810146C</td>
			<td>Rhodamine tagged lipid</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroformation set up</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L glass capillary ringcaps</td>
			<td>Hirschmann</td>
			<td>9600110</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>62.1612</td>
			<td></td>
		</tr>
		<tr>
			<td>H Grease</td>
			<td>Apiezon</td>
			<td>Apiezon H Grease</td>
			<td>Silicon-free grease</td>
		</tr>
		<tr>
			<td>Indium tin oxide coated glass slides</td>
			<td>Sigma-Aldrich</td>
			<td>703184</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>AN2138R1</td>
			<td>0.8 x 38 mm</td>
		</tr>
		<tr>
			<td>Ohmmeter (Multimeter)</td>
			<td>Voltcraft</td>
			<td>VC140</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>VWR</td>
			<td>28676.297</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltage generator</td>
			<td>Keysight</td>
			<td>33210A</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtention of giant unilamellar hybrid vesicles by electroformation and measurement of their mechanical properties by micropipette aspiration

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.

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...

Watch this Scientific Journal Video about Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration at JoVE.com

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

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

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

This protocol facilitates a reliable measurement of the membrane mechanical properties of synthetic and real polymer lipid vesicle using a micropipette aspiration technique. This is the only technique that allows membrane flexibility and instability to be stretched to be assessed in single experiments. This protocol involves a series of procedures from vesicle preparation to the mechanical assessment.It is very important to be patient and to solve any technical issues as they arise. Visualization on these techniques is critical for understanding how to properly treat the capillary surface and how to perform the mandatory prestress step. Showing up the vesicle without defects.Demonstrating procedure will be Martin Fauquignon, a PhD student from the laboratory working on the development of hybrid polymer lipid vesicles. Before beginning the procedure place cold glass micropipette capillaries vertically into holders. And lower the holders until the tips are immersed in freshly prepared glucose bovine serum albumin solution overnight.By the next morning the solution should have risen about one centimeter into the tips by capillary action. Using a 500 microliter glass syringe, equipped with a flexible fused silica capillary, fill each pipette with the glucose solution. Then aspirate the solution from each pipette, before refilling the pipettes with fresh glucose solution several times until all of the serum as been removed.To prepare an electroformation chamber, first clean ITO slides with an appropriate organic solvent, and identify the conductive surface with an Ohmeter. Attach electrical wires onto the conductive side of each slide with adhesive tape. Dip one capillary into ampivial solution until about 5 microliters of the solution has been collected by capillary action.Place the loaded capillary into contact with the center of one glass ITO plate and gently spread the solution across the slide. When the solvent has completely evaporated, apply the solution two more times as just demonstrated. before adding a layer of silicon-free grease on both sides of the opened O-ring spacer around the area of deposition.Next, place the conductive face of a second ITO glass plate on the top of the spacer, and place the electroformation chamber under vacuum for 3 hours to remove any traces of organic solvent. For the electroformation of Giant Unilamellar Vesicles, Plug the electric wires into the generator. Set the generator frequency to 10 Hertz, and the amplitude to 2 Volts, peak to peak.When the voltage has been set, use a syringe equipped with an 0.8 millimeter inner diameter needle to inject one milliliter of 0.1 molar sucrose solution into the chamber, and leave the chamber under the applied voltage and frequency for 75 minutes. At the end of the electroformation, turn off the generator. And use the one milliliter syringe to aspirate a small volume of solution until an air bubble is produced inside the chamber.Tilt the chamber slightly to move the bubble into the chamber, and to help the vesicles detach from the slide surface. And aspirate the entire volume of solution into the syringe. Then, transfer the vesicle solution into a one milliliter plastic tube.To set up the materials for the micromanipulation, aspirate to create a water flow, a tank of pure water, to one holder. And lightly tap while raising the tank to eliminate any air bubbles, and to create positive pressure. Fill a serum coated capillary with fresh glucose solution until a drop forms at the tip.Remove the syringe tubing from the metal holder to create a slight water flow at the end of the holder, and turn the capillary upside-down to connect the glucose drop to the water flow. Then, screw the capillary and the holder together. To position the pipette, glue two glass slides from a custom-made aluminum stage together with vacuum grease.And install the slides onto a microscope stage. Using a one milliliter pipette, form a meniscus between the two slides with 0.1 molar glucose. Place the pipette and its holder on the motor unit of the micromanipulator.Tighten the clamping knob, and use the control panel joystick in coarse mode. Lower the micropipette near the glucose meniscus. Use the fine mode to adjust the position of the tip to the center of the meniscus.Immerse the tip in the glucose to clean its outer and inner surfaces. After a few minutes, withdraw the capillary from the meniscus and replace the glucose with a fresh meniscus. Aspirate two microliters of Giant Unilamellar Vesicles in 0.1 molar sucrose, into a 20 milliliter micropipette tip.Introduce the vesicles into the meniscus. Use the microscope to observe the vesicles at the bottom of the slide chamber. When the vesicles are slightly deflated, reinsert the suction pipette, and focus on the tip of the pipette.Then set the baseline height of the water tank to the pressure at which the motion of the particles is stopped. Surround the meniscus with mineral oil to prevent evaporation. To perform a micropipette aspiration experiment, lower the pipette tip into the meniscus.Create a small amount of suction pressure to aspirate a vesicle. The membrane of the selected vesicle should slightly fluctuate and should not present any visible defects. Use the micromanipulator to raise the pipette to a higher level to isolate the aspirated vesicle from other vesicles.Lower the water tank to approximately 10 centimeters to prestress the vesicle before raising the tank to return the pressure to the initial value. From a height of 0.5 centimeters, slowly decrease the suction pressure until a pressure at which the membrane fluctuates is reached. Then increase the pressure to clearly visualize a tongue in the tip, the projection length of a few microns.To determine the bending modulus, increase the suction pressure, one micrometer at a time in a stepwise manner. Until 0.5 to 0.8 milliNewtons per meter is reached. Waiting five seconds, and taking a snapshot of the tongue after each step.To determine the area compressibility modulus, and lysis tension and strain continue to increase the suction pressure from 0.5 milliNewtons per meter until the rupture tension is reached. In this representative experiment the area compressibility modulus and lysis strain for POPC were in perfect agreement with that expected from the literature. In this table typical values for the polymersomes obtained can be observed.Note that the toughness of the membrane obtained from diblock copolymers is far greater than those obtained from triblock copolymer. Interestingly, using the diblock copolymer, it is possible to obtain a Giant Hybrid Unilamilar Lipid Polymer Vesicles that demonstrate a more robust toughness than that measured for liposomes. This protocol can be helpful for measuring vesicle with a pipette, for example, to measure the permeability of the membrane through asmatic shock.Be sure to complete each step with rigor and precision, especially when setting up the tribune connection. This technique has been exploited to understand physical origin of membrane fission, through the measurement of line tension at the main boundaries in modified vesicles.

Research

JoVE Journal

Chemistry

Fabricating Nanogaps by Nanoskiving

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties

We demonstrate a novel synthesis strategy for the preparation of Pr-doped SrTiO3 ceramics via a combination of solid state reaction and spark plasma ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis and Characterization of Fe-doped Aluminosilicate Nanotubes with Enhanced Electron Conductive Properties

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

TiO2-coated Hollow Glass Microspheres with Superhydrophobic and High IR-reflective Properties Synthesized by a Soft-chemistry Method

TiO2-coated Hollow Glass Microspheres with Superhydrophobic and High IR-reflective Properties Synthesized by a Soft-chemistry Method

This manuscript proposes a soft-chemistry method to develop superhydrophobic and highly IR-reflective hollow glass microspheres (HGM). The anatase TiO2 ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...