Liposome Stock Solution

Before you begin, make sure you have an aliquot of the lipids master mix.

Dry lipid stocks are dissolved in chloroform. Most laboratory plastics are susceptible to chloroform and care must be taken to avoid all plastic labware when working with chloroform solutions. All stocks are stored in glass vials with Teflon-sealed caps. Lipids are transferred using glass Hamilton syringes with Teflon plungers that have been pre-rinsed thoroughly with chloroform.

Once prepared, the lipid solution is stable at 4˚C for a few weeks. Over time, the small unilamellar vesicles formed during initial sonication slowly fuse to make larger, more stable structures. Aged liposome stocks yield patchy bilayers with substantially reduced fluidity and poor surface blocking properties.

Contents

1. Buffers
2. Procedure

1. Buffers

Lipids buffer:

• 10 mM Tris-HCl, pH 8
• 100 mM NaCl
• Filter through a 0.22 μm syringe filter and store at room temperature.

2. Procedure

1. Rinse a new 2 ml glass vial with water and ethanol. Dry the vial in a vacuum oven at 120˚C for about 20 min.
2. Warm the lipid master-stock solutions (in chloroform) to room temperature. Do not open a cold lipids solution to avoid condensing water into the lipids.
3. Rinse a glass Hamilton syringe three times with chloroform.
4. Transfer 220 µl of the lipids master mix into the glass vial using the Hamilton Syringe.
5. This mixture of lipids is evaporated by carefully blowing a weak stream of nitrogen gas while rotating the vial continuously to form a thin, uniform layer of dried lipid on the side walls.
6. The glass vial is then placed in a vacuum dessicator overnight to ensure complete removal of all residual chloroform. Do not place dessicated lipids in a vacuum oven as they will degrade when heated.
7. Add 2 ml of lipids buffer to the dried lipid mixture and allow it to hydrate for at least 2 h to overnight.
8. Vortex the hydrated lipid mixture for 2–3 min to form large multilamellar vesicles. At this point, the liposomes should appear as an opaque, cloudy white solution.
9. Transfer the liposome solution to a 5 ml polypropylene culture tube for sonication. Sonicate the liposomes using a probe tip sonicator to form small unilamellar vesicles under the following settings: (VirSonic or Fisher 1/8” microtip sonicator) set the power output to 15%. Sonicate for 1.5 min with 2-min intervals on ice. Repeat this cycle two more times. After sonication, the liposome solution should clear considerably and become translucent.
10. Filter the liposome solution using a 0.22 μm nylon syringe filter into 1.5 ml Eppendorf tubes. Store the liposome solution at 4◦C for up to 2 weeks. Do not freeze. The final concentration of liposome solution is 10 mg/ml DOPC with 0.5% (w/w) biotinylated DOPE and 8% (w/w) mPEG(550) DOPE.