Biomedical Engineering Reference
In-Depth Information
vesicles. The final increase in thickness following vesicle fusion
amounts to 2.0-2.5 nm, as expected for a lipid monolayer, provid-
ed the vesicle concentration is not too high.
14
When forming planar lipid monolayers and bilayers by vesicle
fusion, the problem of vesicle mere adsorption or partial fusion on
the substrate should be considered. When vesicles fuse spontane-
ously on a support, some of them may remain only partially fused,
or even intact in the adsorbed state. When the surface density of
vesicles is sufficiently high, their presence is revealed by an anom-
alously high thickness attained by the lipid film after vesicle fu-
sion, as monitored by SPR. This phenomenon is particularly evi-
dent with supports consisting of mixtures, with comparable molar
ratios, of two different molecules exposing to the bulk aqueous
phase hydrophobic alkyl chains and hydrophilic functional groups,
respectively.
6,27
With very high vesicle concentrations (5 mg/mL),
monolayer thicknesses greater than 8 nm were reported on hydro-
phobic surfaces.
14
Thus, fusion of small unilamellar vesicles
(SUVs) onto a binary mixture of hydrophobic cholesteryl-
terminated molecules and hydrophilic 6-mercaptohexanol mole-
cules yields AFM images showing heightened areas; their diameter
being close to that of the SUVs denotes the presence of adsorbed
vesicles.
4
The presence of a membrane protein, such as
cyto-
chrome bo3
, in these vesicles increases the number density and the
size of the heightened structures ascribable to adsorbed vesicles.
Analogously, fusion of large unilamellar vesicles (LUVs) on a
hydrophobic support exposing dipalmitoylphosphatidylethanola-
mine (DPPE) alkyl chains to the aqueous phase yields tapping-
mode AFM (TM-AFM) images with a number of dome-shaped
structures.
28
Some of these structures, whose diameter is close to
that of the LUVs, tend to disappear after one hour, due to vesicle
complete fusion. However, if the vesicles contain the membrane
protein Na,K-ATPase, all the domelike structures are stable even
after three hours. In general, adsorption of proteoliposomes (name-
ly, vesicles incorporating membrane proteins) on hydrophobic
surfaces prevents their complete spreading and fusion, due to the
presence of protein molecules with extramembrane domains and to
the hydrophilicity of the outer polar heads of the proteolipo-
somes.
15,29
The presence of heightened areas in metal-supported lipid
films formed by fusion of vesicles labeled with a fluorophore can
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