Self-adhesion among phospholipid vesicles

Article abstract of DOI:10.1021/ja057151s

A compound was synthesized that binds to a phospholipid bilayer via a hydrophobic steroid thereby projecting a strong multi-hydrogen bonding unit into the surrounding water. As shown by light scattering, light microscopy, and cryo-HRSEM, this latter unit self-adheres and induces membrane-membrane attachments, as found in many biological systems. Copyright

Full text of DOI:10.1021/ja057151s

Published on Web 01/11/2006
Self-Adhesion among Phospholipid Vesicles
F. M. Menger* and Hailing Zhang
Department of Chemistry, Emory UniVersity, Atlanta, Georgia 30322
Received October 20, 2005; E-mail: menger@emory.edu
We have previously quipped that “life is a sticky business”...that
located the Upy unit that, as mentioned, hydrogen bonds with itself.
Separating the two moieties is a hydrophilic spacer that can guide
a vesicle-bound Upy into the water. The synthesis of 2 is given in
substrate and enzyme, antigen and antibody, tRNA and mRNA,
leukocyte and endothelial cell, bacteriophage and bacterium, and
1
1
13
sperm and egg are all pairs that stick to each other. The occasion
Scheme 1. Both 1 and 2 were characterized by H/ C NMR,
for this remark was a study of giant vesicles that, via appropriately
selected lipids or polymeric coatings, can be induced to adhere
electrostatically. Stickiness among bilayers also occurs when one
HRMS, and EA following chromatography on silica.
1
Scheme 1. Synthesis of Compound 2
vesicle population contains a donor while a second population
contains an acceptor as with the following pairs: (a) adenine/
2
3
uridine-substituted lipids; (b) positive dendrimer/negative lipid;
4
and (c) barbituric acid-substituted lipids/2,4,6-triaminopyridine- or
5
adenine-substituted lipids. Vesicles have likewise been made to
6
7-9
cluster when ferrous ion or avidin is added to bilayers containing
a terpyridine ligand or biotinylated lipid, respectively. Even DNA
can bind to zwitterionic vesicles and promote their aggregation.10
Specific membrane-membrane interaction that controls self-
assembly and models cell-cell adhesion is clearly an active field
11
of bioorganic chemistry and biophysics.
Many studies of molecular recognition among membranes are
based on two dissimilar vesicles. We, on the other hand, will
describe vesicle adhesion as generated by a single membrane-bound
adhesive agent (1 or 2, drawn below). All our vesicles, in other
Vesicles were prepared by dissolving 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine (POPC) (16 mg) in 2 mL of chloroform.
Evaporating the solvent by rotary evaporator under reduced pressure
created a lipid film that was hydrated via a 10 min stirring with 10
mL of purified water. The resulting suspension was then extruded
1
9 times, back and forth, through a 100 nm pore Whatman
14
Nuclepore membrane to form vesicles with an average hydrody-
namic diameter of 70 nm as revealed by dynamic light scattering
(data collected for 10 min).
When POPC was admixed with compound 1 (95: 5 w/w) prior
words, were identical in composition. Adhesion depended on the
to film formation, we found it impossible to extrude the suspension
through the 100 nm membrane. Applying considerable manual force
to the syringes served only to cause the apparatus to leak. This
was our first indication that compound 1 could indeed cross-link
the bilayers and thereby impede flow through the membrane pores.
We next added solid 1 to a suspension of preformed POPC vesicles,
but 1 was not solubilized. Moreover, 1 was too insoluble in water-
miscible solvents to permit the addition of 1 in a small amount of
cosolvent to the POPC vesicles. This turn of events led us to
synthesize 2, which, with its longer and more hydrophilic spacer,
dissolves in water at sufficient concentrations to admix with
preformed POPC vesicles. Upon mixing the aqueous components,
the opalescence of the vesicle suspensions became immediately
apparent. Figure 1 shows the increase in turbidity at 400 nm as
various amounts of 2 were added to 1 mM vesicular POPC. Since
the total amount of light scattering is proportional to the mass of
remarkable ability of the 2-ureido-4[1H]-pyrimidinone unit (“Upy”)
1
2
in 1 and 2 to dimerize via four strong hydrogen bonds. Thus, its
7
association constant in chloroform saturated with water is 1 × 10
-1 12
M
. Of course, the association constant in pure water, where all
our vesicle experiments were carried out, should be less favorable
than this. On the other hand, vesicles whose surface is covered
with Upy units have the advantage with regard to their stickiness
of being polyvalent. Multiple simultaneous interactions are well-
known to enhance the associative process in both chemistry and
biology.13 And because our vesicles were comprised of only a single
adhesive agent, inter- vs intravesicular competition for hydrogen
bonding sites could be explored.
Adhesive agents 1 and 2 have three distinct sections: At one
end lies a cholesterol derivative whose hydrophobicity serves to
embed the molecules into the vesicle bilayer. At the other end is
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J. AM. CHEM. SOC. 2006, 128, 1414-1415
10.1021/ja057151s CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. Cryo-HRSEM images of extruded POPC vesicles with (A) no
added 2 and (B) 10% added 2.
Figure 1. Change in UV scattering at 400 nm as various percentages of 2
were added to 1 mM POPC vesicles.
and B, respectively. Cryo methods were necessary so as not to
involve water evaporation at high vacuum that would artifactually
promote aggregation. The adhesive nature of the vesicles containing
2
is again evident. Adhered vesicles were also found using 30% 2.
It may be that the much greater curvature of the small extruded
vesicles (and hence a greater Upy separation), relative to that of
the giant vesicles, accounts for the difference in the ability to engage
in intravesicular association.
In summary, we have synthesized a compound that binds to a
phospholipid bilayer via a hydrophobic steroid thereby projecting
a strong multi-hydrogen bonding unit into the surrounding water.
As shown by light scattering, light microscopy, and cryo-HRSEM,
this latter unit self-adheres and induces membrane-membrane
attachments, as found in many biological systems.
Figure 2. Light microscope pictures (phase contrast) of giant vesicles
composed of (A) POPC + 5% 2 and (B) POPC + 10% 2. The vesicles
were formed on a Pt wire where a lipid film was subjected to 100 mV-10
V and 3-10 Hz.
Acknowledgment. This work was supported by the National
Institutes of Health. We thank Dr. Victor Seredyuk and Dr. Robert
Apkarian (Emory IM&MF) for the light and electron microscope
pictures, respectively.
Supporting Information Available: Synthetic details for 2. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
Figure 3. Schematic of intravesicular hydrogen bonding. Orange cone )
steroid (inserted into a bilayer); blue rectangle ) Upy. The average Upy/
bilayer distance has not been established.
(1) Menger, F. M.; Seredyuk, V. A.; Yaroslavov, A. A. Angew. Chem., Int.
Ed. 2002, 41, 1350.
(
2) Berti, D.; Baglioni, P.; Bonaccio, S.; Barsacchi-Bo, G.; Luisi, P. L. J.
Phys. Chem. B 1998, 102, 303.
the particles squared,15 this provided strong evidence that 2 was
indeed functioning as an adhesive agent. It remained only to secure
light and electron microscopy pictures to confirm this conclusion.
Giant vesicles, 30-250 µm in diameter and composed of POPC
plus 5-30% of 2, were prepared by an electroformation method
described previously.16 As seen in Figure 2, adhesions occur at 5%
(
3) Sideratou, Z.; Foundis, J.; Tsiourvas, D.; Nezis, I. P.; Papadimas, G.;
Paleos, C. M. Langmuir 2002, 18, 5036.
(4) Marchi-Artzner, V.; Gulik-Krzywicki, T.; Guedeau-Boudeville, M.-A.;
Gosse, C.; Sanderson, J. M.; Dedieu, J.-C.; Lehn, J.-M. ChemPhysChem
2001, 2, 367.
(5) Paleos, C. M.; Sideratou, Z.; Tsiourvas, D. J. Phys. Chem. 1996, 100,
13898.
(
6) Constable, E. C.; Meier, W.; Nardin. C.; Mundwiler, S. Chem. Commun.
1999, 1483.
2
and, to an even greater extent, at 10% 2. But they are less common
(
7) Chiruvolou, S.; Walker, S.; Israelachvili, J.; Schmitt, F. J.; Leckband, D.;
Zasadzinski, J. A. Science 1994, 264, 1753.
at 20% 2 and totally absent at 30% 2. The simplest explanation is
that when the vesicle loading reaches a level above 20%, pairs of
adhesive agent are sufficiently close together that intravesicular
hydrogen bonding (Figure 3) becomes feasible. Assuming a random
distribution of the adhesive agent with a cross-sectional area equal
to that of POPC, simple geometric considerations show mean
distances between adhesion molecules of 4.3, 3.1, 2.1, and 1.7 nm
at 2/POPC percentages of 5, 10, 20, and 30, respectively. A 1.7
nm intermolecular distance at the vesicle surface apparently allows
an intravesicular association that competes successfully with
intervesicular adhesion.
(
(
8) Noppi-Simpson, D. A.; Needham, D. Biophys. J. 1996, 70, 1391.
9) Vermette, P.; Taylor, S.; Dunstan, D.; Meagher, L. Langmuir 2002, 18,
505.
(
10) Wu, C.-M.; Chen, H.-L.; Liou, W.; Lin, T.-L.; Jeng, U.-S. Biomacromol-
ecules 2004, 5, 2324.
(
11) Weikl, T. R.; Groves, J. T.; Lipowsky, R. Europhys. Lett. 2002, 59, 916.
12) S o¨ ntjens, S. H. M.; Sijbesma, R. P.; van Genderen, M. H. P.; Meijer, E.
W. J. Am. Chem. Soc. 2000, 122, 7487.
(
(13) Menger, F. M.; Bian, J.; Sizova, E.; Martinson, D. E.; Seredyuk, V. A.
Org. Lett. 2004, 6, 261.
(14) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W.; Mayhew, E.; Paphad-
jopoulos, D. Biochim. Biophys. Acta 1980, 601, 559.
(
15) Shaw, D. J. Introduction to Colloid and Surface Chemistry; Butterworth:
London, 1996.
(16) Menger, F. M.; Angelova, M. I. Acc. Chem. ReV. 1998, 31, 789.
Cryo-high-resolution scanning electron microscope pictures of
POPC vesicles without 2 and with 10% 2 are shown in Figure 4A
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