Dope Liposome

Preparation of Cationic Liposomes & Tranfection of Cells Equipment & Materials

Materials

N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)
[1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP)
3β[N-(N’, N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol)
1,2-dioleoyl-sn-Glycero-3-phosphatidylethanolamine (DOPE) Avanti Cat. #850725

 Cationic lipid

Chloroform

Distilled water

Buffer

Glass vials with teflon liners

Glass syringes

Nitrogen or Argon gas

Vacuum system 0.22µm filter (optional)

Procedure

Dry-film method (DF)

• Disslove cationic and neutral lipids (total lipids were 20 umol) in 5 ml chloroform.
• Evaporate the solution for about 50 min at 45 oC.
• After the dried film is formed, use N2 gas to remove the residuary solvent.
• hydrate the dried film with deionized water.
• After sufficient hydration, resuspend the film by vortexing.
• Sonicate the liposomes in deionized water to a final volume of 10 ml

1. Dissolve each lipid component (e.g., DOTAP/DOPE) in chloroform to a convenient working concentration (1-10mg/mL). Aliquot the desired amount of each component into a glass vial using a glass syringe
2. Thoroughly mix the components in the glass vial.
3. Carefully evaporate the chloroform (with adequate ventilation) using a nitrogen or argon stream.
4. Place the lipid residue on a vacuum pump for 10-15 minutes to remove any residual organic solvent.
5. Remove the vial from the vacuum pump and immediately suspend in distilled water at twice the final lipid concentration.
6. Bath sonicate the lipid dispersion to clarity (2-5 min).
7. Add an equal volume of buffer (e.g., 308mM NaCl, 40mM Hepes, pH7.4), and sonicate further for 2 minutes.
8. Solution may be passed through a 0.22µm filter to sterilize.
   

Reverse phase evaporation method (RPE)

• Dissolve a lipid mixture of DC-Chol/DOPE (20 umol in total) in chloroform
• Add deionized water to the lipid solution to form an emulsification automatically.
• Dry the organic solvent under vacuum through attaching to a rotary evaporator.
  
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Ethanol injection method (EI)

The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method.

dissolve lipids (20 umol in total) in 4 ml ethanol
remove ethanol in rotary evaporator leaving behind about 2 ml solution.
Add a constant volume of deionized water to the ethanol solution.
Liposomes formed spontaneously after further evaporation of the residual solvent.
Sonicate liposome suspension using a ultrasonic probe.
Filter resulting liposomes through a 0.22 um sterile filter with the final concentration of DC-Chol was 2 mg/ml.
Store the liposome at 4 ◦ C.

Preparation of Lipid/DNA Mixtures

Combine cationic lipid dispersion with DNA (1µg per 10µg lipid) in a suitable container. Incubate lipid/DNA mixture for 5 min. at room temperature. After 5 min., mixture is ready for transfection of cells.

Transfection of Cells

Wash cell monolayers with HEPES-buffered saline two times. Incubate cells with lipid/DNA mixture in HEPES-buffered saline (3 ml mixture per 100 mm culture dish) at 37°C for 90 min. After the incubation period, either replace the medium or supplement as necessary. Culture cells for desired period of time and harvest.

Notes

Common molar ratios of DOPE:cationic lipid are 3:1 and 1:1. In some cases, a lipid system composed entirely of cationic lipid has been used to transfect cells. Buffer system cited is intended for in vitro use and does not suggest that this system may be suitable for in vivo use.

DOPE-polymer liposome

Mix 100 μl of lipidic polyamine solutions and 100 μl of dioleoylphasphotidyl ethanolamine (DOPE, 50 mg/ml in chloroform, Avanti Polar Lipids, Alabaster, Ala.) in a glass test tube

Remove organic solvents by N2 stream followed by vacuum (rotary evaporator).

A thin lipid film will form on the test tube.

Add 10 ml of distilled water to the test tube

Vortex to suspend the lipid film to give liposomes.

Store the liposome at +4° C