Article

pubs.acs.org/Biomac

Single Lipid Bilayer Deposition on Polymer Surfaces Using Bicelles

Qasim Saleem,†,‡,§ Zhenfu Zhang,‡,§,∥ Amy Petretic,‡ Claudiu C. Gradinaru,*,‡,∥
and Peter M. Macdonald*,†,‡
Departments of †Chemistry, ∥Physics, and ‡Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga
Road North, Mississauga, Ontario, Canada L5L 1C6
*
S Supporting Information

ABSTRACT: A lipid bilayer was deposited on a 3 μm

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diameter polystyrene (PS) bead via hydrophobic anchoring of

bicelles containing oxyamine-bearing cholesteric moieties
Downloaded via UNIV FED DO RIO DE JANERIO on August 5, 2023 at 15:24:15 (UTC).

reacting with the aldehyde functionalized bead surface.

Discoidal bicelles were formed by mixing dimyristoylphospha-
tidylcholine (DMPC), dihexanoylphosphatidylcholine
(DHPC), dimyristoyltrimethylammonium propane
(DMTAP), and the oxyamine-terminated cholesterol deriva-
tive, cholest-5-en-3β-oxy-oct-3,6-oxa-an-8-oxyamine (CHOL-
OA), in the molar ratio DMPC/DHCP/DMTAP/CHOLOA
(1/0.5/0.01/0.05) in water. Upon exposure to aldehyde-bearing PS beads, a stable single lipid bilayer coating rapidly formed at
the bead surface. Fluorescence recovery after photobleaching demonstrated that the deposited lipids fused into an encapsulating
lipid bilayer. Electrospray ionization mass spectrometry showed that the short chain lipid DHPC was entirely absent from the PS
adherent lipid coating. Fluorescence quenching measurements proved that the coating was a single lipid bilayer. The bicelle
coating method is thus simple and robust, can be modified to include membrane-associated species, and can be adapted to coat
any number of different surfaces.

■ INTRODUCTION
Supported bilayer membranes (SBM) were introduced by
McConnell and co-workers three decades ago1,2 and are used
now in an ever-widening range of applications.3−5 SBM consist
of a phospholipid bilayer deposited onto a planar solid
substrate, such as glass or mica, which provides enhanced
mechanical stability while retaining the essential fluid properties
of natural membranes. A polymer “cushion” layer may be
placed between the substrate and the inner leaflet of the
supported bilayer to provide an aqueous space between the
two.6,7 Recently, interest has grown in lipid membranes
supported on colloidal particles,8 since these have many
potential applications as membrane models, in biomolecule
screening, as drug delivery reservoirs, and as therapeutic
vectors. Recently, lipid bilayers supported on silica beads have
even proved useful in the isolation of functional presynaptic
complexes.9,10
Lipid bilayer deposition onto colloidal particles typically
involves adhesion of lipid bilayer vesicles onto the surface
followed by fusion of individual vesicles. Adhesion is
encouraged via electrostatic attraction,11−13 or bioconjuga-
tion,14−16 or hydrophobic attraction between vesicle and
surface.17−20 Fusion into a planar bilayer is induced using
some trigger such as Ca2+ addition,21−23 freeze−thaw,20,24 or
dehydration−rehydration cycling.25 However, such fusion
methods often leave intact liposomes adsorbed alongside or
atop patches of fused lipid bilayer.26,27 A further problem is that
the method of inducing fusion may compromise membrane
protein native structure and function.
Most recently, a new method of lipid bilayer deposition at
solid surfaces has been reported involving the use of
bicelles.28−30 Bicelles, or bilayered micelles, are biomimetic
model membranes consisting of mixtures of long-chain and
short-chain amphiphiles. The long-chain amphiphiles assemble
into a planar lipid bilayer stabilized at its edges by the short-
chain amphiphiles. The planar bilayer region of a bicelle
provides an excellent approximation of the natural membrane
environment, so that bicelles have become increasingly popular
in membrane protein structural studies using a variety of
techniques, including NMR,31,32 EPR,33−36 and X-ray diffrac-
tion.37 Bicelles are also used as membrane protein38 and
pharmaceutical delivery vehicles.39 Relative to liposomes,
bicelles enjoy two important advantages as precursors for
lipid bilayer coatings. First, bicelle fabrication is simple and
carried out under mild conditions conducive to retention of
native membrane proteins structure and function. Second,
bicelles in the form of small planar discs can adapt readily to
rough or irregular surfaces.
Here, we demonstrate that bicelles, when deposited at the
surface of a polystyrene (PS) bead (3 μm diameter) and
retained there by hydrophobic anchors, spontaneously fuse into
a continuous, unilamellar lipid bilayer completely coating and
encapsulating the PS bead. The bicelles were composed of
mixtures of the zwitterionic long-chain phospholipid 1,2-
Received: January 12, 2015
Revised: February 9, 2015
Published: February 9, 2015

© 2015 American Chemical Society 1032 DOI: 10.1021/acs.biomac.5b00042

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dimyristoyl-sn-glycero-3-phosphocholine (DMPC), the zwitter-
ionic short-chain phospholipid 1,2-dihexanoyl-sn-glycero-3-
phosphocholine (DHPC), the cationic long-chain amphiphile
1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP),
and the oxyamine-bearing cholesterol derivative cholest-5-en-
3β-oxy-oct-3,6-oxa-an-8-oxyamine (CHOLOA). The structures
of all four amphiphiles are shown in Figure 1. The molar ratio
Figure 1. Structures of DMPC, DHPC, DMTAP, and CHOLOA.
of long-chain to short-chain species, q = (DMPC + DMTAP +
CHOLOA)/DHPC ≈ 2.1 was chosen to provide small
discoidal bicelles. The oxyamine moiety of CHOLOA is
intended to react with aldehyde groups displayed at the surface
of the PS bead and thus form a covalent oxime linkage to the
cholesterol ring intercalated within the bicelle bilayer, thereby
hydrophobically anchoring the bicelle to the bead surface.
Using a combination of fluorescence recovery after photo-
bleaching (FRAP), fluorescence quenching, and mass spec-
trometry, we show that simply mixing such bicelles with PS
beads at a temperature above the gel-to-liquid-crystalline phase
transition (TM) of DMPC sufficed to produce fusion of
individual hydrophobically anchored bicellar discs into a
continuous, unilamellar lipid bilayer completely encapsulating
the PS bead.
■
EXPERIMENTAL SECTION
Materials. DMPC, DMTAP, DHPC, and NBD-PE (1,2-dioleoyl-
sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-
yl)) were purchased from Avanti Polar Lipids (Alabaster, AL). RhB-PE
(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl)) was purchased from Invitrogen (Carlsbad,
CA). PS latex beads, surfactant-free, ultraclean grade, 3 μm mean
diameter, specific surface area 1.8 × 104 cm2/g, with a high density of
surface-grafted aldehyde (surface density 1/50 Å2) and sulfate charge
groups (surface density 1/190 Å2), were purchased from Life
Technologies (Burlington, ON) and used as received. All other
reagents were purchased from Sigma-Aldrich (Oakville, ON).
CHOLOA Synthesis. Scheme 1 shows the synthetic route to
cholest-5-en-3β-oxy-oct-3,6-oxa-an-8-oxyamine (CHOLOA), com-
mencing from cholest-5-en-3β-oxy-oct-3,6-oxa-an-8-ol (1), which was
prepared as described by Davis and Szoka.40 The n-hydroxyphthali-
mide-activated intermediate (2) was prepared by first dissolving 1.3 g
of 1, 1.4 g of triphenylphosphine (2 equiv), and 0.8 g of N-
hydroxyphthalimide (2 equiv) in 45 mL of anhydrous THF. Upon full
dissolution of the reactants under N2, a dilute solution of diisopropyl
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Scheme 1. Reaction Scheme Outlining Synthesis of the
Cholesterol Bearing Oxyamine Linker
azodicarboxylate (1 mL in 8 mL of anhydrous THF) was added in
dropwise fashion and allowed to react for 16 h. After quenching by the
addition of 20 mL of ethanol, the reaction mixture was concentrated
by rotary evaporation, 70 mL of hexanes was added, the resulting
precipitate of triphenylphosphine oxide (TPPO) was removed by
filtration, and the solution was concentrated again. The residue was
treated multiple times with ethyl acetate (ETOAc) and hexanes to
eliminate any traces of TPPO. The product was applied to a silica gel
column using dichloromethane (DCM) and eluted with a 10−50%
ETOAc/hexanes gradient. After concentration via rotary evaporation,
the product was eluted a second time with a 25−50% ETOAc/hexanes
gradient to yield 1.2 g (72% yield) of a gummy paste. 1H NMR (400
MHz, CDCl3): 7.84 (dd, J = 5.4, 3.1 Hz, 2H), 7.75 (dd, J = 5.4, 3.0 Hz,
2H), 5.38−5.28 (m, 1H), 4.38 (dd, J = 5.6, 3.4 Hz, 2H), 3.91−3.84
(m, 2H), 3.67 (dd, J = 5.8, 3.7 Hz, 2H), 3.61−3.51 (m, 6H), 3.23−
3.06 (m, 1H), 2.39−2.13 (m, 2H), 2.05−1.75 (m, 5H), 1.61−1.24 (m,
15H), 1.19−0.83 (m, 22H), 0.67 (s, 3H). TLC (1:1 EtOAc/hexanes);
Rf = 0.4 (developed with I2 or visualized on Fluorescent TLC plates).
HRMS (ESI+; M + CH3OH adduct + Na+): Calcd, 718.47; found,
718.44
CHOLOA (3) was prepared by reacting 1.2 g of 2 with 0.4 mL (5
equiv) of hydrazine monohydrate codissolved in 20 mL of dry DCM.
Although the expected phthalhydrazide precipitate formed immedi-
ately, the reaction was allowed to proceed for 18 h, after which the
solution was filtered and diluted to 50 mL using chloroform. The
organic phase was washed 5× with water, followed by a single wash
with brine, and was subsequently dried using MgSO4. After filtration
and evaporation of the solvent, 0.7 g (77% yield) of a gum was
obtained. 1H NMR (400 MHz, CDCl3): 5.52 (s, 2H), 5.38−5.27 (m,
1H), 3.92−3.80 (m, 2H), 3.72−3.61 (m, 9H), 3.26−3.11 (m, 1H),
2.42−2.15 (m, 2H), 2.05−1.75 (m, 6H), 1.61−1.24 (m, 13H), 1.19−
0.83 (m, 22H), 0.67 (s, 3H). HRMS (ESI+; M + Na+) Calcd, 556.43;
found, 556.42.
Preparation of Discoidal Bicelles. Bicelles were prepared with a
molar composition DMPC/DMTAP/CHOLOA/DHPC = 1.0/0.01/
0.05/0.5, producing a long-chain/short chain molar ratio q = (DMPC
+ DMTAP + CHOLOA)/DHPC ≈ 2.1, which is expected to yield
discoidal bicelles with diameters in the region of 200 Å.41 Appropriate
quantities of the individual amphiphiles were codissolved in chloro-
form and the solvent was removed under a stream of nitrogen gas. For
FRAP measurements 0.05 mol % RhB-PE was included, while for
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fluorescence quenching experiments, 0.10 mol % NBD-PE was
incorporated. The resulting lipid film was placed in a desiccator
under vacuum overnight to remove final traces of solvent. The lipid
film was hydrated with 1 mL Milli-Q water to produce a lipid
concentration of CL = 1.31% w/v, and the resulting lipid suspension
was subjected to four freeze−thaw cycles wherein the hydrated lipids
were frozen in liquid N2, thawed in a 40 °C water bath, and vortexed.
The bicelle preparation was then stored at 4 °C until use.
Preparation of Unilamellar Liposomes. Unilamellar lipid
vesicles of molar composition DMPC/DMTAP/CHOLOA = 1.0/
0.01/0.05 (note the absence of DHPC) were prepared as described
above for bicelles, except that the multilamellar vesicles formed upon
hydrating the lipid film were extruded 11 times through a 50 nm pore-
size polycarbonate membrane at 37 °C to produce unilamellar vesicles.
Coating of Polystyrene Beads with Bicelles or Vesicles.
Typically 1.0 mg of PS beads were added to 2.0 mg of lipid, either
bicelles or liposomes, and diluted to 2 mL with Milli-Q water. This
represents an approximately 200-fold excess over the amount of lipid
estimated to be required to coat the PS beads with a single lipid
bilayer. Details of this calculation are provided in the Supporting
Information (SI). The PS bead + lipid mixture was gently swirled for 4
h at 30 °C. Excess lipid was removed by low-speed centrifugation (3
min, 6000 rpm) to pellet the lipid-coated beads, which were then
resuspended in 1 mL of water. This washing procedure was repeated
3−5 times.
Fluorescence Imaging and FRAP Measurements. Wide-field
fluorescence images were obtained using a custom-built total internal
reflection fluorescence (TIRF) microscope.42 Confocal fluorescence
imaging and FRAP measurements were carried out using a custom-
built confocal fluorescence microscope.20,43 Lipid-coated PS beads,
labeled with 0.05 mol % RhB-PE, were incubated on a plasma-cleaned
coverslip for 10 min at 20 °C. Confocal images of the sample were
obtained at 20 °C using laser excitation at 532 nm with an intensity of
1 W/cm2. For FRAP experiments, the laser intensity was set to 3 kW/
cm2 for 0.5 s in order to photobleach the lipids within a diffraction-
limited spot on the surface of the PS beads. The photobleached area
constituted around 4% of the total surface area of a lipid-coated PS
bead. Subsequent images were obtained by rescanning the entire bead
with a laser intensity of 1 W/cm2. To obtain high-resolution
fluorescence recovery curves, the photobleached spot was monitored
immediately after the photobleaching using a laser intensity of a 1 W/
cm2 and 10 ms binning. The operation sequence was realized by an
automatic filter wheel (Pacific Scientific, Model No. 5240) controlled
by a custom-written Labview program. In order to reduce the impact
of additional photobleaching, a time lapse acquisition scheme was
employed, with 1 s sampled every 5 s at time delays longer than 1 min
after the photobleaching event. More details about the time-lapse data
acquisition scheme are provided in the SI. FRAP measurements were
repeated on several individual beads and produced essentially identical
recovery curves, as shown in Figure S2.
To extract diffusion coefficients from measured FRAP curves, a
Monte Carlo simulation of the random Brownian motion of
fluorophores on the surface of a sphere was used as previously
described20 and as detailed further in the SI. Briefly, the simulation
uses as input parameters two lateral diffusion coefficients (“fast” and
“slow”) and their relative populations and so generates a FRAP curve
with a specified time resolution and duration. The input diffusion
coefficients and relative populations were varied iteratively to obtain
the best match to the experimental FRAP curves. Details regarding the
parameter search procedure are given in the SI.
For comparative purposes with the three-dimensional geometry of
the sphere, an analytical expression was derived describing two-
dimensional diffusion on a circular disc into a circular photobleached
spot. Details of the derivation are provided in the SI.
Fluorescence Quenching Assay Using Sodium Dithionite. A
sodium dithionite NBD-PE quenching assay was performed on lipid-
coated beads, all incorporating 0.10 mol % NBD-PE. A 1 M sodium
dithionite, 1 M Tris solution was prepared immediately prior to use.
Using a QuantaMaster PTI spectrofluorimeter (Photon Technology
International, Lawrenceville, NJ) equipped with a Quantum North-
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west TC 125 temperature controller (Liberty Lake, WA), the
excitation was set to 470 nm and the emission was monitored at
532 nm using a time based scan via the FelixGX software. An aliquot
of sample was diluted to 2 mL using Milli-Q water in a 4 mL quartz
cuvette and allowed to thermally equilibrate to a set temperature for at
least 10 min. The emission was then monitored for several minutes at
the rate of 1 point/sec. After obtaining a stable fluorescence reading
for 1 min, 20 μL of the sodium dithionite solution was added. The
fluorescence emission intensity was monitored until a new baseline
was achieved, following which 20 μL of 5 wt % Triton X-100 solution
(Triton) was added to disrupt the lipid bilayer and quench the
fluorescence entirely.
Electrospray Ionization Mass Spectrometry (ESI-MS). The
lipid composition of the PS bead bicelle lipid coating was analyzed via
ESI-MS, and compared to whole bicelles. Bicelle lipid-coated PS beads
were stripped of bound lipids by immersion in hexane/isopropanol
(2/1), the naked beads were removed by centrifugation, and the
supernatant was diluted with methanol prior to injection of an aliquot
into the mass spectrometer. Whole bicelles were treated identically.
Using a syringe pump, samples were introduced into a Micromass ZQ
single quadruple electrospray ionization mass spectrometer (Waters
Corporation, MA) operating in the positive ion mode at a flow rate of
30 μL/min with a capillary charge of +3.1 kV, cone voltage of 20 V,
and a source temperature of 100 °C. The samples were scanned over
the range of 430−710 m/z, with a scan acquired every second for 1
min.
■
RESULTS AND DISCUSSION
Chemoselective Bicelle Binding to Polystyrene Beads.
With the objective of depositing a lipid bilayer onto the surface
of a spherical PS bead, we assembled bicelles containing an
oxyamine-derivatized cholesterol moiety, CHOLOA, targeted
to react with aldehyde groups present at the PS bead surface, as
outlined in Figure 2. The oxime linkages formed by reaction
between the oxyamine and the aldehyde functional groups are
an example of chemoselective ligation, referring to the
formation of bonds exclusively between particular pairs of
functional reactants.44 The oxime linkage, in particular, is
chemically stable and forms rapidly under mild conditions
appropriate to biologically relevant applications.45 The
cholesterol moiety of CHOLOA intercalates between the
phospholipids of the bicellar bilayer, ensuring that the
oxyamine group remains anchored to the bicelle surface. The
ethylene oxide spacer ensures that the oxyamine functional
group is minimally sterically restricted and able to encounter
and react with the PS bead surface aldehydes. The presence of
the cationic amphiphile DMTAP within the bicelles encourages
their close apposition with the anionic PS surface via
Coulombic attraction to the sulfate groups present there.
However, electrostatic attraction alone is not sufficient to
ensure a robust lipid coating. But once the oxime link forms,
the cholesterol moiety anchored in the bicelle’s hydrophobic
interior ensures that the bicelles remain attached at the PS bead
surface.
Figure 3A shows a representative wide-field fluorescence
image of PS beads coated with bicelles containing 0.05 mol %
RhB-PE. The cross-section of fluorescence intensity through
one such bicelle-coated PS bead is shown in Figure 3B. The
bead diameter is around 3 μm, as expected. It is evident that
extensive lipid binding has been achieved, even after numerous
washing cycles to remove unbound excess bicelles, and that
lipid has deposited exclusively at the surface of the PS beads.
The near homogeneous density of fluorescence intensity
suggests that defects larger than the diffraction limit (184
nm) do not exist in the deposited layer and that the surface is
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Figure 2. Schematic of bicelles binding to PS bead and their fusion to
form a unilamellar lipid bilayer. Discoidal bicelles are composed of
mixtures of DMPC (orange head groups) and DHPC (green head
groups) with a small percentage of CHOLOA (red oblong) and
DMTAP (not indicated). Initial attraction between bicelles (cationic)
and PS beads (anionic) is electrostatic. Oxyamine groups of
CHOLOA, displayed at the bicelle surface, react with aldehyde groups
displayed at the PS bead surface to form oxime linkages, thereby
anchoring the bicelles to the PS beads. DHPC is preferentially
removed by washing, which forces fusion of adjacent bicelles into a
continuous unilamellar lipid bilayer covering the PS bead.
uniformly coated. However, the resolution limits in such images
do not permit one to draw conclusions regarding the thickness
of the lipid coating, that is, single or multiple bilayer, or
whether individual bicelles have fused into a continuous bilayer
and formed a complete permeability barrier. Nor can it be
assumed that the composition of bound amphiphiles is the
same as that of the added bicelles.
DHPC is Absent from the PS Bead Lipid Coating. To
examine qualitatively the composition of the lipids coating the
PS beads, ESI-MS analysis was undertaken. ESI-MS is rapid,
sensitive, and readily discriminates species having different
molecular weights, such as DHPC and DMPC,46,47 but rather
similar 31P and 1H NMR chemical shifts. Results obtained with
control bicelles are shown in Figure 4A versus lipids stripped
from the PS bead coating in Figure 4B. Since the ESI-MS
instrumental response increases with decreasing phospholipid
acyl chain length,46 DHPC is detected with far greater
sensitivity than either DMPC, or DMTAP, or CHOLOA.
(Control measurements on DMPC/DHPC mixtures of known
composition confirm this to be the case, data not shown). In
Figure 4A, specifically, the ESI mass spectrum of bicelles of
molar composition DMPC/DMTAP/CHOLOA/DHPC =
1.0/0.01/0.05/0.5, q ≈ 2.1, shows DHPC peaks at 454.26 m/
z [MDHPC + H+] and 476.23 m/z [MDHPC + Na+] that appear
with far greater intensity than those due to DMPC (678.46 m/z
[MDMPC + H+], 700.43 m/z [MDMPC + Na+]) or CHOLOA
(MCHOLOA + CH3OH + Na+ = 588.29 m/z). DMTAP (554.49
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Figure 3. (a) Wide field fluorescence image of bicelle-coated PS beads
containing 0.05 mol % RhB-PE and obtained at 20 °C. The scale bar
represents 5 μm. (b) Cross-section of fluorescence intensity across an
individual bicelle-coated PS bead.
m/z), being present at only 1% relative to DMPC, is barely
detected. In the ESI mass spectrum of the lipids bound to, and
then stripped from, the PS bead surface, shown in Figure 4B,
peaks corresponding to DHPC are conspicuously absent, and
only DMPC and DMTAP are evident. Thus, DHPC appears to
have been removed preferentially during the various washing
stages to which the PS bead lipid coatings were subjected
during fabrication. Morigaki et al.30 in their studies of POPC/
DHPC bicelles coating glass surfaces also suggested that DHPC
was absent from the deposited bilayer.
It is noteworthy that no CHOLOA peak is evident in the ESI
mass spectrum of lipids stripped from the PS bead lipid coating
(Figure 4B), implying that all CHOLOA present in the bound
lipid fraction had reacted with surface aldehydes and become
covalently attached through the oxime linkage. The PS bead
surface density of aldehydes is ∼50 Å2/CHO. The bicelle
surface density of CHOLOA, assuming 60 Å2 per DMPC and
given 5 mol % CHOLOA relative to DMPC, is roughly 1200
Å2/CHOLOA. Thus, it is reasonable that all CHOLOA within
a bicellar disc bound at the PS bead surface had become
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Figure 4. ESI mass spectrum of (a) bicelles having molar composition
DMPC/DMTAP/CHOLOA/DHPC = 1.0/0.01/0.05/0.5, q = 2.1;
(b) Lipids stripped from PS beads after binding via oxyamine-aldehyde
chemoselective formation of oxime linkages.
covalently attached and, hence, could not be removed merely
by stripping with organic solvent.
The DMTAP peak in Figure 4B is notably intense,
particularly so in comparison to Figure 4A, implying that
DMTAP is enriched at the PS bead surface relative to the 1 mol
% DMTAP present in the initial bicelle formulation. The PS
bead surface density of sulfates is ∼190 Å2/SO4, which is
roughly a factor of 30 greater anionic charge density than the
bicelle cationic surface density due to 1 mol % DMTAP.
Bicelles are highly fluctional, in that there is a constant dynamic
exchange of lipids between individual discoidal fragments, as
established from fluorescence studies.48 Thus, it is entirely
plausible, given the higher surface charge density of the PS
beads versus the bicellar discs plus the fluctional nature of
bicelles, that DMTAP becomes enriched at the PS bead surface
via exchange under the motive force of Coulombic attraction.
Because ESI-MS, as shown in Figure 4A,B, is not strictly
quantitative without extensive calibration efforts, further direct
quantitative measurements will be necessary to address this
possibility.
Bicelles Bound at the PS Bead Surface Fuse into a
Continuous Lipid Bilayer. To address the question of
whether the bicelles bound at the PS bead surface fuse into a
continuous lipid bilayer, FRAP measurements were performed
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at room temperature (20 °C). Figure 5 shows the results
obtained for the case of bicelles allowed to bind PS beads at 30
Figure 5. FRAP images (measured at 20 °C) of 0.05 mol % RhB-PE-
labeled PS bead bicelle lipid coatings formed at 30 °C: prior to
photobleaching (a), immediately after photobleaching (b), and 20 min
subsequent to photobleaching (c).
°C, that is, above the TM of DMPC. Prior to photobleaching
(Figure 5A), the RhB-PE fluorescence is a continuous ring
arising from lipid bound at the PS bead surface. Immediately
after photobleaching a diffraction-limited spot, the ring of
fluorescence is discontinuous (Figure 5B), but it reacquires the
continuous shape on a time scale of 10−20 min (Figure 5C).
This can only occur if individual bicelles have fused and formed
a continuous lipid bilayer (or bilayers) on the surface of the PS
bead. A movie showing the time course of the recovery of the
continuity of the ring of fluorescence was made by stringing
together a sequential series of confocal images of a photo-
bleached PS bead and is provided in the SI.
To quantify the lateral diffusion of lipids bound to the PS
bead surface, FRAP curves were measured and analyzed using
Monte Carlo simulations to extract lateral diffusion coefficients.
Figure 6 shows the experimental FRAP curve obtained at 20 °C
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Biomacromolecules Article

population, supported bilayer membranes generally exhibit two

roughly equal lipid populations with differing lateral diffusion
properties and this is usually attributed to friction between the
inner bilayer leaflet and the underlying support that is absent
from the outer leaflet.50,51 Even if the bilayer is separated from
the support by spacer groups intended to alleviate such friction,
the anchors are immobile and can introduce a “picket fence”-
type frictional barrier within the inner leaflet.19,20
The mobile fraction of lipids in the supported bilayer m
(Table 1) is extracted by applying eqs 1 and 2 to the raw FRAP
data:
(1 − m)(1 − B) + m(1 − B) = F(0) (1)

(1 − m)(1 − B) + m[(1 − fa ) + fa (1 − B)] = F( +∞)

(2)
where 0 ≤ B ≤ 1 is the degree of photobleaching in the
photobleached area, fa is the fraction of total fluorophores
photobleached, and F(0) and F(+∞) are the intensities
measured immediately after the photobleaching event (average
of first 10 data points) and at the end of the recovery (average
of last 30 data points), respectively, both normalized relative to
the prephotobleaching intensity F(−∞).
For bicelle-coated PS beads, the mobile lipid fraction is on
the order of 70% (Table 1). This must be considered a lower
limit, since an inspection of Figure 6 shows that the
fluorescence continues to recover slowly out to long times.
In order to examine the possibility that the biphasic FRAP
curves were merely the result of a single population diffusing
Figure 6. FRAP recovery curves (black), Monte Carlo simulations across the three-dimensional geometry of the PS beads, as
(red) and 2D analytical plot (blue) for 0.05 mol % RhB-PE-labeled PS opposed to two distinct populations, a two-dimensional system
bead bicelle lipid coatings. Simulations assumed two different lateral was modeled, consisting of a disk of finite radius with a small
diffusion coefficients and population weightings. Results of best-fits for photobleached spot. The derivation of the analytical formula
the lateral diffusion coefficients and populations are listed in Table 1. describing FRAP curves in this instance is provided in the SI
Parameters used in 2D analytical plot are identical to those used in the
(eq S3). Figure 6 shows the fluorescence recovery in the two-
simulation. Also shown are FRAP recovery curves for PS bead lipid
coatings formed from liposomes of molar composition DMPC/ dimensional disk case using the same diffusion coefficients and
DMTAP/CHOLOA = 1.0/0.01/0.05. populations obtained from the Monte Carlo simulation for the
three-dimensional sphere. The comparison shows that while
from a single PS bead bicelle lipid coating prepared at 30 °C, FRAP in the “fast” phase is similar in the two cases, recovery in
along with the corresponding best-fit Monte Carlo simulation. the “slow” phase is enhanced in the three-dimensional case.
Essentially identical recoveries were obtained when different This is likely due to the more complex geometry of the
beads were samples, as detailed in Figure S2. Lateral diffusion photobleached region in the three-dimensional case,20 relative
coefficients and relative populations obtained from the Monte to the strictly circular photobleached spot assumed for the two-
Carlo simulations are listed in Table 1. The simulation indicates dimensional case. Nevertheless, this comparison makes clear
that the dimensionality of the PS bead is not the origin of the
Table 1. Results of Monte Carlo Simulations of FRAP biphasic FRAP curves, but rather, that there are indeed two
Curves of Lipids Bound to PS Beads different diffusing populations.
Permeability of PS Bead Lipid Bilayers. To examine the
mobile integrity of the lipid bilayer coating, NBD-PE fluorescence
lipid D1 D2 fraction
morphology −14
(×10 m2/s) −16
(×10 m2/s) P(D1)/P(D2) (%) quenching by sodium dithionite was examined. Ideally,
dithionite ions added externally will not permeate to the
bicelles 1.0 4.6 50:50 68 ± 3
interior compartment of an intact unilamellar spherical lipid
liposomes 0.40 1.4 40:60 46 ± 7
bilayer, and the NBD-PE fluorescence would be reduced to
50% of the initial value. When PS bead lipid coatings formed
the presence of two populations, virtually equal in size, but from bicelles were interrogated in this fashion, as shown in
differing by several orders of magnitude in their lateral Figure 7, NBD-PE fluorescence was reduced to roughly 30%,
diffusivity. Simulations assuming a single population of diffusing indicating significant permeation of dithionite to the interior
lipids always produced inferior fits to the experimental recovery side of the lipid bilayer coating.
curves. Comparisons of several single-population versus two- Increased permeability is a general concern with SBM and
population simulations are provided in Figure S3. perturbations by the underlying supporting surface are often
The lateral diffusion coefficient of the “fast” fraction (D = 1 × implicated as the cause. For instance, Nollert et al.52 found that
10−14 m2 s−1) is in accord with values for DMPC lipid bilayers an intact POPC lipid bilayer deposited on a hard glass surface
at temperatures below TM = 24 °C.49 As for the “slow” (planar or spherical) was freely permeable to dithionite ions, an
1037 DOI: 10.1021/acs.biomac.5b00042
Biomacromolecules 2015, 16, 1032−1039
Biomacromolecules
Figure 7. Sodium dithionite induced quenching of NBD-PE
fluorescence from PS bead lipid coatings formed using bicelles
(black curve) of molar composition DMPC/DMTAP/CHOLOA/
DHPC = 1.0/0.01/0.05/0.5, q ≈ 2.1 and liposomes (gray curve)
formed of molar composition DMPC/DMTAP/CHOLOA = 1.0/
0.01/0.05. Measurements were performed at 15 °C. Dithionite was
added at time point “D” and the detergent Triton X100 was added at
time point “T”.
effect attributed to surface roughness. Similarly, Ng et al.18
found that coating soft hydrophobically modified dimethacry-
lamide beads with egg PC resulted in a continuous bilayer that
was almost completely permeable to cobalt ions, in contrast to
the impermeability of egg PC liposomes. It was proposed that
the stress experienced by the bilayer when in close proximity to
the dynamic, possibly highly corrugated, surface of the bead was
the origin of the increased permeability.
Comparison with Liposome-Based PS Bead Coatings.
Because liposomes are most commonly used to deposit lipid
bilayer coatings on solid supports, comparative studies were
undertaken for the case of PS bead lipid coatings formed with
CHOLOA-containing liposomes. Fluorescence images were
largely indistinguishable from those obtained with bicelle-based
coatings (not shown). At the level of resolution achievable with
the technique used it could not be determined from such
images whether individual intact liposomes were bound at the
PS bead surface or if fusion into regions of planar bilayer had
occurred or some mixture of the two. However, FRAP recovery
curves, as shown in Figure 6, were characterized by a large
immobile fraction and much slower apparent lateral diffusion
coefficients than the corresponding bicelle-based coatings, as
detailed in Table 1. Furthermore, fluorescence quenching
measurements, shown in Figure 7, revealed fluorescence
reduced to about 60% of the initial value, in line with
reductions measured on the initial liposomes. Given these
FRAP and fluorescence quenching results, it would seem that,
in the absence of any external trigger, fusion of the liposomes
bound at the PS bead surface was incomplete. This might be
the result of electrostatic repulsion between adjacent cationic
liposomes.
Mechanism of Single Bilayer Formation from Bicelles
Bound at the PS Bead Surface. The mechanism by which a
single bilayer coating forms at the PS bead surface upon
deposition of bicelles, as deduced from the experiments
described here, is summarized schematically in Figure 2. The
initial interaction is electrostatic attraction from a distance
between cationic bicelles and the anionic PS bead surface.
Upon close approach, the oxyamine groups displayed at the
bicelle surface are able to chemoselectively react with aldehyde
groups displayed at the PS bead surface, forming oxime linkages
1038
Article
that anchor the bicelles via the hydrophobically intercalated
cholesterol ring. The neutral, relatively water-soluble short-
chain species DHPC is preferentially removed during the
washing steps to remove excess lipid. Elimination of DHPC
forces fusion of bicelles into larger, unilamellar structures, as the
self-assemblies seek to minimize edge regions formerly
occupied by DHPC. Provided a sufficient density of surface-
bound bicelles had been attained initially, fusion occurs and
begets a continuous unilamellar lipid bilayer completely
encasing, and closely conforming to, the PS bead surface.
■ CONCLUSIONS
A novel method has been described for the deposition of a
single lipid bilayer onto a hard polymer bead starting from
discoidal bicelles and using chemoselective chemistry to
hydrophobically anchor the lipid assemblies. This method of
lipid bilayer deposition is of general relevance for coating both
hard and soft matter systems, extending the established use of
bicelles in coating silicon28,29 and lipidic30 surfaces. Since
discoidal bicelles can be made with a variety of saturated and
unsaturated phospholipids, various lipid bilayer coating
compositions can be achieved. Relative to conditions required
for liposome fusion into a continuous lipid bilayer, bicelle
fusion is induced simply and spontaneously under mild
conditions. And because bicelles can be composed to optimize
membrane protein native structure and function, this approach
should prove advantageous in depositing membrane proteins at
such surfaces for analytical, diagnostic, or therapeutic
applications.
■
ASSOCIATED CONTENT
*
S Supporting Information
Calculation of polystyrene bead−bicelle surface ratios, compar-
ison of continuous and time-lapse FRAP data acquisition
modes, the reproducibility of the lipid bilayer coating on PS
beads, Monte Carlo FRAP simulation, 2D vs 3D FRAP
recovery analysis, 1H NMR spectra of compounds, and an avi
movie showing the time course of the recovery of fluorescence
recovery. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: claudiu.gradinaru@utoronto.ca.
*E-mail: pm.macdonald@utoronto.ca.
Author Contributions
§
These authors contributed equally to this work (Q.S. and
Z.Z.).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Financial support from the Natural Science and Engineering
Research Council (NSERC) of Canada is acknowledged
(P.M.M and C.C.G). Q.S. was supported by an Ontario
Graduate Studies (OGS) Scholarship and Z.Z. was supported
by a Canada Institutes of Health Research (CIHR) Training
Grant. We would like to thank University of Toronto Profs.
Ulrich Krull, David McMillen, and Heiko Heerklotz for kind
access to instruments.
DOI: 10.1021/acs.biomac.5b00042
Biomacromolecules 2015, 16, 1032−1039
Biomacromolecules Article

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1039 DOI: 10.1021/acs.biomac.5b00042

Biomacromolecules 2015, 16, 1032−1039