Encapsulation: Phase Transfer

Overview¶

Emulsion phase transfer is a general method for encapsulating an aqueous inner solution (e.g., Cytosol) inside a lipid bilayer to form liposomes. In this protocol, you will prepare your lipid mixture, form a water-in-oil emulsion from your inner solution and lipid mixture, and transfer that emulsion across an oil–water interface into an outer solution, yielding liposomes.

Each Module specification gives its own reference composition, outer solution requirements, and expected behavior once encapsulated. This page covers the encapsulation process given those parameters.

Materials and Equipment¶

Protocol¶

Prepare Lipids in Mineral Oil¶

• Add 1 mL of mineral oil to the glass vial using a 1 mL pipette.

• Add the lipids in the above Table to the glass vial on top of the mineral oil using the appropriate glass syringe.

• Briefly vortex the lipid-oil mixture for 5 s to mix.

• Evaporate the chloroform from the lipid–oil mixture:

  • Place glass vial in a 55 °C dry bath in a fume hood.

  • Shield with aluminum foil to protect from light.

  • Evaporate uncovered for 4 h.

• Meanwhile, clean your glass syringe.

  • Add 4 mL of chloroform to a glass bottle using a glass 10 mL serological pipette.

  • Rinse your syringe with that chloroform 5 times.

  • Dry the syringes with the plunger removed inside the fume hood for 3 h to 4 h to allow remaining chloroform to evaporate.

• Cool the lipid-oil mixture to room temperature (between 10 min and 15 min). Your lipid-oil mixture is now ready for use.

• Return the plungers to the syringes and store them in their designated location.

• Dispose of chloroform waste following applicable chemical safety guidelines.

Assemble Outer Solutions¶

• Prepare 1.5 mL microcentrifuge tubes labeled with the appropriate reaction.

• Prepare outer solution by mixing glucose stock solution and water to reach your target osmolarity:

Encapsulate Inner Solution¶

• Allow lipid-oil mixture to come to room temperature as you perform these next steps.

• For each liposome encapsulation, set up a 1.5 mL tube rack with two 1.5 mL microcentrifuge tubes. Number the tubes according to the number of reactions assembled. Label the two tubes for each reaction:

  • T—transfer

  • L—liposomes

• Add 300 µL of the appropriate glucose outer solution to each of the tubes labeled T. Vortex thoroughly and spin down.

• Assemble at least 30 µL of your inner solution per reaction (see Assemble Base Cytosol for an example).

• Once at room temperature, add 150 µL of the lipid-oil mixture on top of each assembled inner solution reaction.

• Emulsify the lipid-oil and inner solution reaction by running the tube along a row of empty slots on the 1.5 mL tube rack. Drag your tube across empty slots at least 50 times until you form a stable emulsion with a homogeneous milky appearance.

• Immediately layer each emulsion on top of the outer solution by slowly pipetting down the side of the corresponding T tube.

• Centrifuge T tubes at 9000 g for 10 min at room temperature to pellet the liposomes.

• Extract the liposomes from each T tube:

  • Gently remove the oil layer and lipid debris from the top of each T tube with a 1000 µL pipette.

  • Gently resuspend the pellet by mixing by pipette 10-15 times.

  • Extract liposomes by pipetting 50 µL to 100 µL of pellet and outer solution from T and transfer liposome sample to the respective liposome tube L.

• Hold liposomes on ice until you are prepared to begin measurement.

• Transfer 5 µL liposomes and 20 µL of corresponding outer solution onto a 384-well glass bottom plate (5x dilution). If the density of liposomes appears too high under the microscope, dilute them with outer solution to facilitate data analysis.

• Observe liposomes by fluorescence microscopy.

Downloads¶