Embedding resorcinarene cavitands in lipid vesicles

Article abstract of DOI:10.1039/c2nj20961f

A fluorescently labeled resorcinarene cavitand has been successfully embedded in DLPC lipid vesicles and imaged using confocal microscopy. The cavitand resides exclusively in the bilayer. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012.

Full text of DOI:10.1039/c2nj20961f

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LETTER
Embedding resorcinarene cavitands in lipid vesiclesw
Katie M. Feher, Hai Hoang and Michael P. Schramm*
Received (in Gainesville, FL, USA) 16th November 2011, Accepted 30th January 2012
DOI: 10.1039/c2nj20961f
A fluorescently labeled resorcinarene cavitand has been successfully
embedded in DLPC lipid vesicles and imaged using confocal
microscopy. The cavitand resides exclusively in the bilayer.
difluorodinitrobenzenes. Conversion of the eight nitro groups
to amines followed by in situ condensation with propionyl
chloride gave an octaamide that possessed one free hydroxyl
for further transformation.1,11 We next transformed the alcohol to
a mesylate with mesyl chloride then to azide using sodium azide.
Copper iodide with tris-(benzyltriazolylmethyl)amine (TBTA)
catalyzed triazole formation was straight forward using propargyl
amine appended nitrobenzoxadiazole (NBD) as our fluorophore
coupling partner giving 3 in 64% yield from azide 2 (see ESI for full
experimental detailsw).12 1H NMR and high-resolution mass
spectrometry are fully consistent with the assigned structure.
Having prepared our fluorescent cavitand we next tested its
ability to bind small molecule guests. Mixing 3 with adamantyl
amine in mesitylene-d12 gave rise to characteristic upfield signals
associated with guest encapsulation1 (see ESIw). Similar results
were not seen in aqueous micelles. The reasons are not known at
this time—an analogous tetra ethyl footed cavitand did bind guests
in aqueous micelles.5 The functionality of 3 in water has not been
fully explored, but by changing the feet or rim this issue can be
resolved. Having established the cavitand as an active host in
organic solvents we turned our attention to developing a protocol
for visualization when in an aqueous vesicle environment.
Resorcinarene cavitands are deep, highly ordered supramolecular
species that have a proclivity for small molecule guest binding in
organic solution.1 Alterations to the feet2 and rim3,4 have
afforded water soluble species. More recently, several cavitands
have been shown to retain their function as small molecule
binders5,6 and as pH responsive molecular switches7 while
embedded in aqueous micelles. In addition to micelles, lipid
bilayers have also proven to be compatible; guests appended
with fluorophores and proteins bind to resorcinarenes as
determined by surface plasmon resonance.8 With the cellular
uptake of calixarenes demonstrated,9 resorcinarenes are sure
to follow. Micelles provide an excellent environment for
characterizing host–guest behavior using NMR techniques.
The larger size of vesicles and more ordered lipid bilayer
environment provide additional methods of characterization
and more importantly provide a better model for the cell. In
this report we demonstrate a method suitable for embedding
and visualizing resorcinarene cavitands in the lipid interior of
phosphocholine vesicles. This work sets the stage for visualizing
and quantifying host guest binding events in a lipid bilayer
environment.
To better understand their behavior in lipid bilayer environments
we prepared a fluorescent resorcinarene cavitand. We had several
key criteria in mind with our design: (1) an ideal cavitand should
have significant hydrophobic character so that it partitions into the
bilayer, (2) it should be amenable to fluorescent tagging, and (3)
should retain its ability to serve as a small molecule host after
chemical modification and embedment.
Monofunctionalized resorcin[4]arene 1 was readily prepared
by a literature method from the condensation of resorcinol,
hexanal, and 2,3-dihydrofuran (Fig. 1).10 We then transformed
the shallow resorcinarene into a cavitand through addition of four
California State University Long Beach, Department of Chemistry
and Biochemistry, 1250 Bellflower Blvd., Long Beach, CA 90840,
USA. E-mail: michael.schramm@csulb.edu; Fax: +1 562-985-8557;
Tel: +1 562-985-1866
w Electronic supplementary information (ESI) available: Full
synthetic procedures and characterization of all new compounds is
provided. Additional confocal images and full z-scan analysis is
included. See DOI: 10.1039/c2nj20961f
Fig. 1 Synthesis of fluorescent monoNBD functionalized octaamide
cavitand 3 and MMFF rendition (see ESI for full detailsw).
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Many methods exist for the preparation of giant unilamellar
vesicles (GUVs) that are suitable for optical characteriza-
tion.13 One method that has been underutilized in our opinion
is incredibly flexible and affords polydisperse GUVs in 4
min.14 Characterization of the bilayer of these vesicles is
simple using phase contrast and confocal microscopies.15 In
short this method entails: (1) lipid(s) dissolved in chloroform
and methanol are suspended in an aqueous buffer solution;
(2) desired additives are introduced; (3) the suspension is
rotavapped at 40 1C for 4 min to remove all organic solvents,
and optionally (4) gel permeation or dialysis can be applied to
remove extravesicle additives. The samples are then placed on
inverted deep well microscope slides and examined at 10–40 Â
magnification using confocal microscopy.
migration of the dye into the lipid bilayer (Fig. 3A). Confocal
microscopy z-scan analysis illustrates the location of the dye
when imaged at half the height of the vesicle. To confirm this
result we prepared vesicles and incorporated 1 mole% 1-acyl-
2-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl)-sn-
glycero-3-phosphocholine prior to vesicle formation. Again the
lipid bilayer is fluorescent (Fig. 3B). With cyclohexylamine
NBD we noted that many multilamellar vesciles and other very
small species were formed.
Having established the visual controls necessary to differentiate
between the localization of hydrophilic and hydrophobic fluores-
cent molecules we next examined the behavior of resorcinarene 3.
Cavitand 3 (1 mol%) was introduced to lipid prior to vesicle
formation. Removal of organic solvents resulted in the formation
of viable vesicles. Z-scan analysis at several heights (Fig. 4)
reveals fluorescence exclusively in the lipid bilayer.
Hydrophilic small molecule fluorophores are not expected
to partition into the membrane bilayer of DLPC vesicles. We
prepared 2-(Thioureidofluorescein)ethyltrimethylammonium
Chloride (4) by condensation of fluorescein isothiocyanate
and 2-aminoethyltrimethylammonium chloride following
literature procedure.8 When 4 was added to a solution of DLPC
vesicles, the aqueous exterior is fluorescent and the vesicle
interior is dark (Fig. 2A). When 4 was added to the lipid solution
prior to vesicle formation, fluorescence is observed both inside
and outside of the vesicle and the lipid bilayer is notably darker
(Fig. 2B). Size exclusion chromatography or dialysis removes the
external dye giving dye-loaded vesicles (Fig. 2C). These control
experiments illustrate the behavior of hydrophilic fluorophores—
they reside exclusively in the aqueous environment. The same
results were observed for carboxyfluorescein (See ESIw).
In the first frame we see two vesicles at their apex. Moving 2
microns in the z-dimension the bilayer is revealed, and after
another 2 we arrive at the half-height of the upper vesicle. In
the final frame have arrived at the bottom of the upper vesicle,
but not quite at the lower vesicle, which is larger in size (See ESI
for all frames and corresponding DIC imagesw). This result is
consistent with the results for hydrophobic species seen in Fig. 3.
Cavitand 3 is convincingly located in the lipid bilayer.
We anticipate that most resorcinarene cavitands will have
the same behavior due to their significant hydrophobic surface.
Based on work with lipid bilayers on SPR (surface plasmon
resonance) chips, the water soluble cavitand with ethyl feet and
four carboxylates displays consistent results.8 The incorporation
of resorcinarenes into lipid bilayers and their host guest binding,
along with our report sets the stage to study other pressing issues
with these synthetic receptors. Microscopy is now an additional
tool to study events in these larger ensembles where NMR
spectroscopy is no longer the most easily applicable technique.
The binding of small molecules in micelles,5,6 the binding of
proteins to surfaces with embedded cavitands,8 two component
switching,7 and ion sensing16 are all recent first inroads of
applying resorcinarenes to biological model systems and problems.
We believe that resorcinarene cavitands have even more potential
as controllable optical storage devices, molecular machines and
may provide other exciting opportunities for applications to
Hydrophobic molecules on the other hand would be expected
to behave differently. When cyclohexylamine-NBD was added to
a vesicle solution (before of after vesicle formation) we observe
Fig. 3 Fluorescent confocal images (Olympus Fluoview 1000, IX-81;
ex: 488; em 559; 40 X) of prepared DLPC vesicles that were treated
with 1% cyclohexylamine-NBD (A), and DLPC vesicles that were
prepared with 1% 1-acyl-2-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-
dodecanoyl)-sn-glycero-3-phosphocholine (B). Vesicles are approximately
10 microns. For DIC images see ESI.w
Fig. 2 Fluorescent confocal images (Olympus Fluoview 1000, IX-81;
ex: 488; em 559; 40 Â) of prepared DLPC vesicles that were then
treated with 4 (A), DLPC vesicles that were prepared with 4 present
prior to vesicle formation (B), and that were then subjected to dialysis
(C). Vesicles are approximately 10 microns. For DIC images see ESI.w
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Slides were viewed on an inverted Olympus Fluoview 1000
confocal laser scanning system utilizing a Olympus IX-81
microscope with a 40x oil objective and 2x digital zoom. The
dyes were excited by a 488 nm argon ion laser, and emission
was captured at 559 nm. The dimensions of the images taken
were 1024 Â 1024 pixels and a z-scan analysis was performed
capturing a minimum of 10 planes. On average vesicles imaged
were 10 Æ 2 microns in diameter.
The project described was supported by Grant Number
SC2CA167636 from the National Cancer Institute. The
content is solely the responsibility of the authors and does
not necessarily represent the official views of the National
Cancer Institute or the National Institutes of Health. Confocal
microscopy was supported by a grant from the National
Science Foundation (DBI0722757), with additional support
from the Department of Biological Sciences and College of
Natural Science and Mathematics.
References
Fig. 4 Fluorescent confocal images (Olympus Fluoview 1000, IX-81;
ex: 488; em 559; 40 X; z-scan analysis) of prepared DLPC vesicles that
had 1 mol% of cavitand 3 present at four different heights in the
z-dimension (heights listed). Vesicles are approximately 10 microns.
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Experimental procedure for vesicle preparation and visualization:
1,2 dilauroyl-sn-glycero-3-phosphocholine (15 mL, 0.0804 M in
chloroform, Avantis Polar Lipids), methanol (200 mL),
chloroform (1 mL), and Hepes (3 mL, 0.01 M in 18.2 MO
H2O) were placed in a 50 mL pear-shaped flask. The solution
was slowly rotovapped at 10 mmHg, 40 1C, 40 rpm for
4–5 min.14 After two distillation events were observed an
opaque liquid resulted. When using cavitand 3, 1 mol% was
added prior to vesicle formation. When using fluorescein-
choline 4, 2.5 mol% was added prior to or post vesicle
formation. When using carboxyfluorescein, 5 mol% was
added prior to or post vesicle formation (see ESIw). For gel
filtration we employed Bio Rad Econo-Pacs 10 DG 10 mL
Disposable Chromatography Columns and for dialysis
Spectra/Pors Dialysis Membrane, MWCO: 6–8000, Nominal
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Flat Width: 10 mm, Diameter: 6.4 mm, Vol/Length: 0.32 mL cmÀ1
,
Lot # 3248269, using a minimum of 3 buffer exchanges.
Deepwell slides were prepared using 80 mL of GUV solutions.
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876 New J. Chem., 2012, 36, 874–876
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012