Using the Droplet Transfer Method to Reliably Prepare Giant Unilamellar Vesicles

This beginners' guide presents a general protocol for the preparation of Giant Unilamellar Vesicles (GUVs) via the droplet transfer method, discussing the critical steps and providing insights to help possible adjustments required for different applications, including synthetic biology and microrobotics.

We aim at cell-like microrobots that navigate through body tissues for administering therapies locally. They're based on giant unilamellar vesicles and a phospholipidic membrane and contain active particles. This protocol offers a guide for beginners who want to adopt and adapt the droplet transfer method in synthetic biology and in other fields, for example, in microrobotics.

Compared to other GUV preparation techniques, this protocol offers high encapsulation efficiency and straightforward execution, making it accessible to any research group with basic laboratory equipment. We will assess GUV-based microrobots'navigation in tissue-like settings and implement sensing strategies to enable autonomous movement towards diseased cells. To begin, move the glove chamber either inside or beside a chemical hood.

Open the glove chamber and place a crystallizer filled with silica particles at the bottom of the chamber. Cover the crystallizer with a white support surface, and then layer it with paper towels. Insert all required materials into the glove chamber, and place the glass vial containing the chloroform solution on a cork ring holder or a 150-milliliter beaker positioned as close to the operator's side as possible.

Now, close the glove chamber, ensuring that the two halves are securely aligned and cannot slide. Use the provided plastic screws to completely seal the chamber. Close the outlet valve and submerge pipe number five into a 500-milliliter beaker that is half filled with water.

Next, connect the valve-regulated end of pipe number two to the vacuum line. Then, connect the other end to the nitrogen line using pipe number three. Open the valve and turn on the vacuum line until the gloves inflate and become too rigid to bend.

Then, close both the vacuum line and the valve. Next, open the nitrogen line until the gloves deflate and extend out from the glove chamber. After donning gloves, use a Hamilton syringe to transfer the specified volume of DOPC in chloroform solution into a dark glass vial.

Seal the vial with the cap containing the needles. Now, connect pipe number six to pipe number eight and pipe number seven to pipe number nine using the two-way junctions. Remove the gloves from the glove chamber and open the outlet valve.

Then, open the nitrogen line slowly and monitor the gas flow for bubbles in the beaker. Gradually increase the nitrogen flow until a steady but observable bubbling rate is reached. After three minutes of flow, close the nitrogen line.

Once the final bubble exits the beaker, close the outlet valve. Put the gloves back on and disconnect pipes eight and nine from the two-way junctions. Next, open the vial and move the cap back to the 50-milliliter tube.

Then open the oil bottle and transfer the required amount of oil into the dark vial. Seal the vial with the cap again, remove the gloves, and open the glove chamber. Vortex the sealed lipid suspension vial vigorously for at least 30 seconds.

Then incubate it at 80 degrees Celsius in a water bath for 30 minutes. After vortexing the vial again, incubate it overnight at room temperature. Using scissors, cut off the cap from a five-milliliter tube.

Drill an approximately eight-millimeter hole in the center of the cap to fit a P1000 pipette tip. If the hole is too wide to fit the tip, discard the cap and restart with a new one. With a two-by-five-centimeter strip of sandpaper, smooth in the edges of the drilled hole.

Place the cap back on the five-milliliter tube and centrifuge at 3, 000g for five minutes to eliminate any residual plastic particles. Then, remove the cap and discard the tube. Now, cut the cap from a new five-milliliter tube and replace it with the prepared holed cap.

Insert a P1000 tip through the hole with an O-ring locked between the tip and the cap to prevent further downward movement. Fill the tube with 5.5 milliliters of outer solution through the tip. Vortex the lipid suspension vial prepared earlier for at least 30 seconds.

Then, incubate it in the 50-degrees Celsius water bath for 30 minutes. Allow the water solutions prepared earlier to equilibrate at room temperature for at least 10 minutes. Now, pipette 300 microliters of outer solution into a 1.5-milliliter tube or the previously modified five-milliliter tube.

Slowly layer 100 microliters of lipid suspension on top of the oil solution, ensuring the formation of two distinct and continuous phases. Let the interface stand undisturbed for 10 minutes to equilibrate. In a two-milliliter tube, pipette 250 microliters of lipid suspension, followed by 6.25 microliters of inner solution to prepare a 2.5%water-in-oil emulsion.

Mix the two phases until the emulsion appears homogeneous and stable. Transfer 200 microliters of the prepared emulsion onto the oil solution lipid suspension interface formed earlier. If using a 1.5-milliliter tube, simply close the tube.

If using the modified five-milliliter tube, place a rubber cap on the pipette tip and gently transfer the entire tube into a 50-milliliter conical tube. Centrifuge the tubes at room temperature at appropriate speed. After centrifugation, verify that an opaque pellet is present at the bottom and that the oil phase is clear at the top.

To remove oil from the five-milliliter tube containing GUVs, retrieve the pipette tip from the tube while ensuring the rubber cap remains in place. Discard the tip and retain the rubber cap, O-ring, and hold cap for future use. Instead, if using a 1.5-milliliter tube, use a P1000 micropipette to remove the oil solution, leaving behind approximately 50 microliters and making sure not to disturb the pellet.

Change the pipette tip, then resuspend the pellet in the remaining 50 microliters of the solution. Finally, transfer the resuspended material to a clean two-milliliter tube. Lipid solutions prepared under high humidity conditions produced giant unilamellar vesicles with significantly lower yields and a greater prevalence of disordered aggregates compared to those prepared in a humidity-controlled chamber, although some aggregates were still present in the latter.

At 3, 300g for 20 minutes, the yield and quality of sucrose-loaded vesicles were optimal, whereas both lower and higher centrifugation speeds let to unfavorable outcomes, such as reduced yield or aggregate formation. Mineral oil resulted in vesicles with a larger average diameter distribution, while silicone oil AR 20 generated a significantly higher overall vesicle count.