Biointerfacing Polyelectrolyte

Microcapsules –
A Multifunctional Cargo System
B R I TA N I C O, GY L E LY N E T T E P.
P U N Z A L A N , M A RY D E N I S E K .

BIO192 / E01
Polyelectrolyte multilayer - supported liposomes
• lipid bilayer - coated polyelectrolyte multilayer capsules
• fabricated through the conversion of liposomes into lipid bilayers to cover the
capsule surface in analogy to the cell membrane
Biomimetic membrane fabrication process
• preparing a hollow polyelectrolyte multilayer capsule by the alternating LbL
procedure templated on spherical particles
• PSS and PAH were used as wall components
• The protocol used for the lipid coating was mainly based on electrostatic attraction between
the last polyelectrolyte layer and unilamellar vesicles composed of lipids with opposite
charges
• vesicles were incubated with the capsule dispersion in order to let adsorption occur
• the samples were then centrifuged to remove nonadsorbed lipids
• various spectroscopic and microscopic techniques to evidence the successful coating of lipid
bilayers on the surface of capsules
ζ potential
• is the potential difference between the mobile dispersion medium and the
stationary layer of the dispersion medium attached to the dispersed particle.
• ζ potential change of the surface of polyelectrolyte capsules identified the
effective lipid adsorption.
• The inversion of the charge sign of the capsule surface after contact with oppositely
charged vesicles proved the surface modification by lipids
Lipid adsorption on capsules
• N - (7 - nitro - 2,1,3 - benzoxadiazol - 4 - yl) ( NBD ) - phosphocholine
◦ a fluorescent lipid

• confocal laser scanning microscopy (CLSM)

• presence of a fluorescent ring around capsules and as the fluorescence intensity
distribution was homogeneous, lipid coverage was considered as uniform
• Coating quality quality were analyzed by flow cytometry.
• an intense and thin fluorescence peak was obtained.
Phase Transition Temperature
• used to estimate whether lipid layer on the capsules forms an organized layer
structure or not.
• Differential scanning calorimetry ( DSC )
• measurements can provide information on the phase of the adsorbed lipids by
measuring the phase transition enthalpy
Surface Roughness
• Atomic Force Microscopy (AFM)
• the surface of lipid layer - coated polyelectrolyte capsules is almost perfectly
smooth
• the root mean square of the height variations obtained is less than 1 nm.
• the surface of polyelectrolyte layers prior to the lipid deposition presented a
considerable roughness
• rms value was about 7 nm
Formation of Lipid Bilayer when
adsorbed onto capsules
• freeze - fracture electron microscopy experiment
• bare particles and polyelectrolyte coated particles
• fracture took place through the hydrophobic polystyrene interior.
• assemblies surrounded by lipids
• fractured through the weakest part, which is the mid - plane of the adsorbed lipid bilayer.
Thickness of Lipid Layers
• Förster energy transfer between two fluorescent probes was quantitatively
assessed
• lipid layers were sandwiched between two polycation dye - labeled PAH layers
• Lipid layers marked with fluorescein (donor molecule)
• PAH layers marked with rhodamine (acceptor molecule)
• Considering that the thickness of one polyelectrolyte layer is 1.5 – 2 nm, the
distance between both probes was estimated to be around 4 – 5 nm in the
case of lipid coverage.
Successful coating of lipid layers
• permeability assays were carried out on the lipid/polyelectrolyte capsules
• 6 – CF was incubated with the capsules and observations by CLSM showed that
the lipid envelope decreases the capsule permeability to molecules by a factor
of 10^3
• incorporating 6 - CF to reference capsule solutions (i.e., without lipids) with
partially, or completely lipid - covered capsules
• confocal microscopy showed that the probe entered into reference capsules
with partially lipid – covered capsules
• continuous lipid shell avoided this phenomenon and led to perfectly
impermeable hollow systems.
 Phospholipase A2
• PLA2 was added into a lipid - coated
capsule suspension to tune the
permeability
• stereoselectively hydrolyze the sn-2 ester
linkage of enantiomeric l-phospholipids,
such as l-α- dipalmitoylphosphatidylcholine
( l - DPPC ), to release fatty acids and
lysophospholipids
Formation of Asymmetric
Lipid Bilayers on the
Surface of LbL-Assembled
Capsules
CHAPTER 2.3.2
 A key feature of cell plasma membranes is the asymmetric distribution of
phospholipids, which controls important cellular processes such as signal
transduction and fusion.
 Phosphatidylserine– inner leaflet
 Phosphatidylcholine – outer leaflet
 An “inner” lipid monolayer
Polyelectrolyte microcapsules
from organic solution was
with a narrow size distribution
deposited onto the oppositely
were first fabricated via LbL
charged polyelectrolyte
assembly
microcapsules

An “outer” lipid monolayer

PDDA and PSS were employed
Asymmetric lipid bilayer- coating was formed by
as the polycation and
modified polyelectrolyte incubating small unilamellar
polyanion while DDAB and
capsules or vesicular particles vesicles with the lipid
DHP, sodium salt was used as
were obtained monolayer-coated colloids in
cationic and anionic lipids
water
 Multilayer film formation followed by microelectrophoresis shows the alternating ζ potential
as a function of PSS/PDDA layers and DDAB/DHP asymmetric layers.
 The ζ potential measurements confirmed the assembled system with DHP as the inner leaflet
and DDAB as the outer leaflet.
 The quartz crystal microbalance ( QCM ) was employed to follow the assembly process.
 Fluorescence microscopy was applied to examine the deposition of DDAB and DHP layers onto
PSS/PDDA- coated particles. NBD- dipalmitoylphosphatidylethanolamine ( DPPE ) was
incorporated in the anionic DHP layer as the anionic lipid dye probe.
 In order to prove that the lipid membrane is indeed asymmetric (i.e., that the
inner and outer layers of the bilayer comprise different lipid molecules),
fluorescence quenching assays were carried out to determine the distribution of
probe lipids between the inner and outer leaflets of the bilayer membrane.
The fluorescence emission intensity measurement before and after addition of
Na2S2O4 solution shows that the addition of the quencher to the dispersion
indeed causes a reduction in the emission of only the probe on the outer
monolayer of the membrane.
 Relative fluorescence intensity is a function of fluorescence quenching time
and thus confirmed the formation of an asymmetric bilayer.
The polyelectrolyte multilayers on either side of the asymmetric lipid bilayer
membrane play an important role in preserving the membrane ’s stability.
Assembly of Lipid Bilayers
on Covalently LbL-
Assembled Protein
Capsules
CHAPTER 2.3.3
 The use of covalent bonds to assemble LbL multilayer films can provide significant advantages
compared to traditional electrostatic assembly
 Have high stability due to the covalent bonds formed and therefore do not disassemble with
changes in pH or ionic strength
 The fabrication of Hb capsules could imitate in some sense its structural function in the living
system, and may help us to understand its properties and further make protein – based
biodevices.
 GA is used as a chemical cross - linker because it has less effect on the protein activity.
Poly(ethylenimine) ( PEI ) was first adsorbed on template particles to produce an amino
functionalized surface. Then, the GA and Hb were alternately adsorbed.
 TEM and CLSM – capsule formation. Wall thickens of capsules can be
controlled by the adsorption cycles of alternate GA/Hb
 UV- Vis spectra of GA/Hb – absorption band of heme at 411nm. Hb essentially
remains in the capsules.
 Cyclic voltammetry and potential – controlled amperometric measurements –
cross-linked Hb capsules keep their heme electroactivity and are not denatured
 FITC (Fluorescein isothiocyanate) – permeability of the assembled GA/Hb
microcapsules were tested. GA/Hb, 5 capsules are impermeable to FITC –
dextran with molecular weights 2000 and 500 kDa, while molecular weights
below 70 kDa can partly and even completely permeate into the capsule interior
 PAH/PSS 5 capsules, the Hb protein shells have selective permeability. The
permeability decreases with the increase of wall thickens.
 As phosphatidylcholine lipid is a major component of biological membranes,
egg phosphatidylcholine was chosen as a lipid model to cover the capsules, but
a small fraction of negatively charged lipid, phosphatidic acid, was added in
order to promote the adsorption and fusion of vesicles.
 The existence and stability of the lipid bilayer was proved by means of CLSM.
 In order to validate the use of lipid bilayer - coated protein multilayer capsules
as a cell membrane model, an ion channel protein, H + - ATP synthase, was
incorporated in the supported lipid bilayer.