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Biophysical Studies of Cell-Penetrating Peptides 227
preparation where amphiphilic lipids are hydrated by an aqueous medium and form
bilayers, which self-close as a particle. Agitation alone (vortexing) results in dispersion
of large multilamellar vesicles (LMV) with an onion-like structure formed by
many lipid bilayers separated by water in layers. By repeated freeze-thawing, the
particle size can be made more uniform. LMV is usually not a suitable model system
for biophysical studies, since there is no single inner compartment. LMVs can be
useful, however, for broad-band nuclear magnetic resonance (NMR) studies ( 2 H, 31 P)
of the molecular order within a membrane
For biophysical studies various unilamellar vesicle preparations are useful model
systems. The vesicle concept should be restricted to those particles with a single,
unilamellar, membrane bilayer enclosing an inner compartment. Depending on the
size of the vesicles, it is common to classify them as small (SUV; diameter 20 to
50 nm), large (LUV; 100 to 200 nm), and giant (GUV; 10 to 100 µm) unilamellar
vesicles. The small vesicles (SUV) have large curvature, causing nonuniform tension.
A larger vesicle should, in principle, better mimic the plasma membrane of a true
biological cell because, with a large inner volume, the interior compartment can be
more easily monitored. However, for optical spectroscopic studies, light scattering
from large particles can be a severe problem. In such studies, SUV samples are
usually preferred.
10.3.1.1 Small Unilamellar Vesicles (SUVs)
Disruption of LMV dispersions by ultrasound (sonication) will produce an SUV
sample. The treatment is rather harsh and should be carried out with cooling and a
protective nitrogen atmosphere. When a probe tip sonicator is employed, metal
particles must be removed by centrifugation. Since the size of the vesicles has some
distribution, ultracentrifugation or chromatography will make a sample more monodisperse.
An SUV is metastable and should be stored only for a few days (above
the transition temperature). Hydrolysis of the lipids may occur during sonication
and the storage. The number of phospholipids required to form a typical SUV is on
the order of 3000, with approximately 60% of the molecules occupying the outer
layer. The large curvature will create a tighter packing of the lipids in the inner leaflet.
10.3.1.2 Large Unilamellar Vesicles (LUVs)
This type of preparation can also be useful for biophysical studies. 17 The simplest
preparation procedure is to freeze-thaw an LMV suspension by several cycles,
followed by extrusion repeatedly through a polycarbonate microfilter with, e.g.,
100-nm pores. LUV samples may be stored in a refrigerator.
10.3.1.3 Giant Unilamellar Vesicles (GUVs)
This type of vesicles is observable by a light microscope and may be manipulated
by microinjection. 18 GUVs can produced from phospholipids dissolved in a chloroform
and methanol mixture. Hydration can take place either after removal of the
solvent, or at the same time as the pressure is reduced to allow rapid evaporation
from a water–organic solvent mixture; however, it is not evident that all the organic