Liposomes for DNA Nanotechnology: Preparation, Properties, and Applications - DNA Nanotechnology: From Structure to Function - page 60

4.4 Preparation of Liposomes

Liposomes are classified according to their size and the number of bilayers. Small
unilamellar vesicles (SUV) are in the size range of 15 to 50 nm, while large
unilamellar vesicles (LUV) can go up to one micron in size. Cryo-transmission elec-
tron microscopy (cryo-TEM) is a powerful technique to observe these liposomes,
where the size distribution and the number of bilayers can be precisely resolved.
In this technique, dispersed liposomes are vitrified on a TEM grid in liquid ethane
to prevent ice formation and to preserve the native liposome structure. On the other
hand, negative staining with heavy metal salts such as ammonium molybdate results
in deformed liposomes but allows for a quick sample preparation. Giant unilamellar
liposomes (GUVs) are greater than a few microns and can be resolved by optical
microscopy.
Various methods have been developed to prepare each type of liposome. In gen-
eral, lipids are dissolved in an organic solvent such as chloroform, which needs to
be fully evaporated. There are several methods to completely dry the lipid including
using rotary evaporator, spray drying, lyophilization, or low-pressure evaporation
for several hours in the presence of a neutral desiccant [ 12 ]. We and many other
groups find that using a gentle nitrogen flow on top of the sample followed by
overnight incubation in a vacuum oven can sufficiently remove chloroform resulting
in a dry lipid film. Hydration of the film must be performed above the lipid T c .Inthe
case of lipid mixtures, the lipids must be thoroughly dissolved in an organic phase
and then dried, whereas rehydration should occur at a higher temperature than the
highest T c in the mixture. To encapsulate drugs or other molecules, hydration should
be carried out in a buffer containing these molecules. Hydration under mechanical
agitation results in formation of micrometer-sized multilamellar vesicles (MLV).
These suspensions appear to be very cloudy due to the large liposome sizes [ 11 ].
In order to transform MLVs into unilamellar vesicles, many methods have
been developed such as sonication, extrusion, detergent depletion, and solvent
injection [ 17 , 19 ]. Two of the methods are summarized here. High-energy sonic
fragmentation is used to generate SUVs. In this case, the MLV suspension is
exposed for several minutes to a titanium tip probe sonicator at a particular
temperature. The possibility of introducing metal contaminants released upon
sonication is one of the disadvantages of this method. To minimize this problem, the
suspension is then filtered using, e.g., 0.45-

m pore size filter to remove residual

titanium particles [ 20 ].
Extrusion, on the other hand, allows the formation of homogenous SUVs and
LUVs with precise size control. In this case, the MLV suspension is forced through
a filter with a defined pore size. Commercial filter membranes ranging from 50 nm
to 1

m are available. To achieve a uniform size distribution, two membranes can
be stacked. It needs to be noted though that extrusion using membranes with a
pore size 200 nm yields a polydispersed suspension of multilamellar liposomes.

Unilamellar liposomes with a narrow size distribution can only be produced with
membranes with a pore size of smaller than 200 nm. Extrusion is typically carried

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