Layer-by-layer Cell

Membrane Assembly
Sandro Matosevic and Brian M. Paegel*
Brian M. Paegel
Ph.D., Chemistry, University of California, Berkeley, 2003
B.S., Chemistry, Duke University, 1998
Assistant Professor, Scripps Research Institute
Introduction
Phospholipid membranes are complex supramolecular assemblies
involved in every aspect of biological function.
Important traits to replicate include curvature, fluidity, lamellarity,
and composition.
Current methods:
Bulk assembly without control over vesicle size, content, membrane
composition, and lamellarity.
Microfluidic vesicle assembly allows control over size variation and can
encapsulate cargo with 80% efficiency.
Deposition based strategy that controls single layer vesicle starting with lipid-
stabilized water-in-oil emulsions.
Method
Droplets of oil are emulsified into water
droplets and then centrifuged.
This makes them cross a oil/water phase boundary.
Lipid droplets are added to each water-in-oil
droplet and stick to it.
This adds a layer of lipids to each droplet every time
the water droplet crosses a phase boundary with the
oil. This adds several layers of lipids and needed
content using a microfluidic droplet array.

Method Cont.
The droplet array uses 3 fluidic input lines.
Oil-lipid mixture of dioleoylphosphatidylcholine
(DOPC) dissolved in squaline
Cytoplasmic aqueous phase of TAE buffer to fill the
vesicles - AQ
cy
Extracellular aqueous phase - AQ
ex
Results
Test unilamellar presence after single
deposition:
Test perfuses haemolysin into cells to act as channels.
Different sized flourescent dyes are added and only
small dye goes into molecule.
Same permeability with vesicles loaded with DNA that
encodes for haemolysin gene.
Haemolysin only permeates single bilayers. Proves
unilamellar structure.
Results Cont.
Test the quantities of material
going into each monolayer
formed during the synthesis of
the membrane.
Add fluorescent reporter lipids into
each monolayer addition to test
quantities.
If added correctly, fluorescence
should increase linearly.
Final cooling and discard of waste
should result in a fraction of final
fluorescence. Inversely proportional
to number of depositions.
Conclusion
Lipid bilayer assembly via deposition is now a viable route to
asymmetric unilamellar and double-bilayer membranes.
Unilamellar vesicles can support functional insertion and pore-
forming protein complexes, and support a basic cell-like metabolism.
References
Sandro Matosevic &Brian M. Paegel. (2013). Layer-by-layer cell
membrane assembly. Nature Chemistry 5, 958963.

Thank You