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

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.