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Procedure for Preparation of Cationic Lipid/Oligomer Complex

Rationale

Normal procedure for preparation of lipid:DNA complex requires suspending the cationic lipid in aqueous buffer and sonication to form small vesicles. The cationic lipid vesicles are mixed with the aqueous DNA solution at a weight ratio of 10:1, lipid:DNA, and incubated for a period of time. During this incubation it is presumed that the positively charged liposomes associate and coat the surface of the DNA, giving the DNA a cationic lipid layer which facilitates interaction with and tranfer through the cell membrane. With small DNA fragments or oligomers such as antisense DNA, the DNA particle is typically much smaller than the lipid particle. In this case, the lipid does not coat the DNA, but rather the DNA coats the surface of the liposome. This would serve to disrupt the critical liposome:cell surface interaction and inhibit transfer through the membrane. To effectively coat these small DNA particles, the lipid needs to be presented to the DNA in its monomer (or small aggregate) form. This can be accomplished by dissolving the lipid in an organic solvent and either dispersing the organic in an aqueous solution of the DNA, or incubating the lipid and DNA together in the organic solvent. The solvent of choice is ethanol since it is both miscible with water and non-toxic to biological systems in the event that residual solvent remains in the suspension.

Procedure A (ethanol lipid solution:aqueous oligomer solution)

  1. Dissolve Cationic Lipid in Eethanol.
    1. If the lipid sample is stored in chloroform, evaporate the chloroform using dry nitrogen or argon and place lipid residue on a vacuum system for 1 hour to remove residual chloroform.
    2. Add ethanol to dry lipid residue and dissolve completely using moderate heat (40-50°C) and sonication if necessary.
    3. Adjust the concentration such that the volume of ethanol lipid solution added to aqueous oligomer solution is 10% of the aqueous solution volume.
  2. Dissolve Oligomer in a Suitable Volume of Aqueous Buffer.
  3. Add Ethanol Lipid Solution to the Aqueous Oligomer Solution at a Weight Ratio of 10:1, Lipid:Oligomer.
    1. Thoroughly mix the lipid:oligomer suspension by vortexing or sonication.
    2. Incubate the suspension for 5-10 minutes to allow lipid:oligomer complex to form.
    3. If residual ethanol is not desired, heat the suspension to 40-50°C and bubble nitrogen through the suspension until ethanol is removed.

Procedure B (ethanol lipid solution:ethanol oligomer solution)

  1. Dissolve Cationic Lipid in Ethanol.
    1. If the lipid sample is stored in chloroform, evaporate the chloroform using dry nitrogen or argon and place lipid residue on a vacuum system for 1 hour to remove residual chloroform.
    2. Add ethanol to dry lipid residue and dissolve completely using moderate heat (40-50°C) and sonication if necessary.
  2. Dissolve Oligomer in a Suitable Volume of Ethanol.
  3. Combine the Ethanol Solutions in a Weight Ratio of 10:1, Lipid:Oligomer.
    1. Thoroughly mix the lipid:oligomer suspension by vortexing or sonication.
    2. Incubate the suspension for 5-10 minutes to allow lipid:oligomer complex to form.
    3. Evaporate the ethanol using dry nitrogen, argon, rotory evaporator, etc., and dry the lipid residue on a vacuum system for 1 hour to remove residual ethanol.
  4. Resuspend Lipid:Oligomer Complex in Aqueous Buffer and Sonicate to Disperse.