Biology Reference
In-Depth Information
to monitor solute leakage can be avoided. For example, carboxyfluorescein (CF
) leakage can
be directly monitored by fluorescence in a cuvette
[44]
. This technique works because at very
high concentration (~160 mM) CF
is self-quenching and appears as dark brown, but at low
concentrations fluoresces a brilliant yellow. CF
is sequestered inside the liposomes at 160
mM where it appears brown and starts to fluoresce yellow as the solute leaks out, where it
first appears at low concentration in the external solution. Therefore liposome leakage is
directly proportional to CF
fluorescence intensity.
In another example, glucose leakage is followed by a linked series of enzymatic reactions
in a cuvette followed by light absorbance at 340 nm
[45]
. Liposomes are made with seques-
tered glucose. The bathing solution contains ATP, Mg
2
þ
, NADP, and the enzymes hexokinase
and glucose-6-phosphate dehydrogenase. As glucose leaks out of the liposomes,
the
following reactions occur in the bathing solution:
ð
Hexokinase
Þ
glucose-
Mg
2þ
Mg
2
þ
Glucose
þ
ATP
þ
6
-phosphate
þ
ADP
þ
/
Glucose
-
6
-
Phosphate Dehydrogenase
Þ
ð
NADP
þ
Glucose-6-Phosphate
þ
6-
Phosphoglucanate
þ
NADPH
/
NADPH absorbs at 340 nm while NADP
þ
does not. Therefore glucose leakage from the
liposomes is proportional to an increase in absorbance of NADPH at 340 nm. Since no
samples are taken for analysis, glucose permeability can be continuously monitored.
Unilamellar Vesicles
The original liposome preparation method of Bangham produces multilamellar vesicles
(MLVs) of varying size
[38]
. MLVs have several important shortcomings that limit their
use. They are not of uniform size and a large portion of their total lipid is sequestered inside
the onion-like vesicles. This severely limits the internal aqueous space available to solutes
and makes it impossible to accurately assess the amount of lipid comprising the external
membrane surface. In addition, evaluating the net leakage rate is very complex as solutes
must traverse several bilayers before making their way through to the external bathing solu-
tion. Therefore a different type of liposome, one having only a single surrounding bilayer and
of uniform size, was required. This type of liposome is generally referred to as a unilamellar
vesicle (ULV) in contrast to a multilamellar vesicle (MLV). ULVs are further subdivided by
size. The following list covers the most common types of liposomes:
Abbreviation
Name
MLV
Multilamellar Vesicle
ULV
Unilamellar Vesicle
SUV
Small Unilamellar Vesicle
LUV
Large Unilamellar Vesicle
GUV
Giant Unilamellar Vesicle
