An ion conductor that recognizes osmotically-stressed phospholipid bilayers

Article abstract of DOI:10.1021/ja026895s

A synthetic ion conductor (1), derived from cholic acid and spermine, has been found capable of recognizing osmotic stress in liposomes made from 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine [(C16:1)PC]. Thus, when large unilamellar vesicles of (C16:1)PC are placed under hypotonic conditions, the Na⁺/Li⁺ transport activity of 1 increases by as much as 1 order of magnitude, relative to isotonic conditions Copyright

Full text of DOI:10.1021/ja026895s

Published on Web 08/27/2002
An Ion Conductor That Recognizes Osmotically-Stressed Phospholipid
Bilayers
Prasun Bandyopadhyay, Punam Bandyopadhyay, and Steven L. Regen*
Department of Chemistry, Lehigh UniVersity, Bethlehem, PennsylVania 18015
Received May 14, 2002
In this work, we show how a synthetic ion conductor is capable
of recognizing a phospholipid bilayer that has been placed under
+
+
osmotic stress. Specifically, we show that the Na /Li transport
activity of a spermine/sulfated-cholic acid conjugate (1), in lipo-
somal membranes derived from 1,2-dipalmitoleoyl-sn-glycero-3-
phosphocholine [(C16:1)PC], increases as the liposomes are
converted from an isotonic to a hypotonic state. The potential for
using osmotic stress in bacterial membranes as a “therapeutic target”
is noted.
2
Figure 1. Plot of kobsd versus (mol % 1) for 200-nm vesicles made from
C16:1)PC at 35 °C under (9) isotonic conditions, and hypotonic conditions
(
having initial osmolarity gradients of (2) 417 and (b) 517 mosM,
respectively.
5 3
Na P O10; the latter two salts function as a shift reagent. Exact
osmolarities were determined experimentally for each salt solution
+
used. These dispersions were then analyzed for Na efflux by use
Previous studies have shown that certain conjugates derived from
23
5
+
+
of Na NMR spectroscopy at 35 °C. Similar dispersions that were
analyzed for Li influx by Li NMR gave pseudo-first-order rate
constants, kobsd, that were identical to those observed for Na efflux.
A plot of kobsd versus (mol % of 1) was found to be linear,
cholic acid and spermine promote Na /Li transport across phos-
pholipid bilayers and that the transport activity can be strongly
dependent upon membrane thickness. For example, the activity of
+
7
+
2
1
in bilayers made from (C16:1)PC is ca. 700 times greater than in
indicating that transport-active dimers are involved (Figure 1). Here,
it is assumed that only a small fraction of the conjugate is
aggregated, where it can be shown that:
bilayers derived from 1,2-dioleoyl-sn-glycero-3-phosphocholine
1
[(C18:1)PC]. On the basis of its ability to recognize small changes
in membrane thickness, it occurred to us that such a compound
might also be able to recognize osmotically stressed bilayers. In
particular, when liposomes are exposed to hypotonic solutions,
osmotic swelling leads to an increase in surface area and to a higher
2
k2[monomer]
kobsd
)
K
2
occupied area per molecule. For neighboring phospholipids to
where K is the equilibrium constant for dissociation of the dimer,
is the rate constant for ion transport, and [monomer] is the
maintain hydrophobic contact, the number of gauche conformers
in the acyl chains must increase, and the bilayer must undergo
thinning. Here, we provide the first evidence for osmotic stress
k
2
analytical concentration of the ion conductor that is present in the
dispersion.6
3
,4
recognition of a phospholipid bilayer by a synthetic ion conductor.
Analogous dispersions that were prepared under two different
+
+
In this study, we examined the activity of 1 for Na /Li transport
in liposomal membranes made from (C16:1)PC under varying
osmotic conditions. Thus, large unilamellar vesicles (200-nm
average diameter) were prepared using standard extrusion methods,
+
+
hypotonic conditions were also examined for Na /Li transport.
In one preparation, the initial osmolarity gradient and osmotic
pressure were 417 mosM and 10 atm, respectively. A second
preparation had an initial gradient and pressure of 517 mosM and
a 450 mM aqueous (10% D
percentages of 1. Gel filtration, via minicolumn centrifugation
prepacked Sephadex G-25, PD-10, Pharmacia Biotech), removed
2
O) NaCl solution and varying mole
13 atm, respectively. An analysis of the 10-atm dispersion showed
ion-transport characteristics that were very similar to those found
under nonstressed conditions, except that the activity was signifi-
(
the NaCl that was present in the external aqueous phase. Subsequent
mixing with appropriate salt solutions afforded liposomal disper-
sions that were osmotically balanced, having an external aqueous
phase that was 440 mM in LiCl, 2.4 mM in DyCl3, and 7.3 mM in
-
1
-2
2
cantly greater; that is k /K increased from 0.0011 min mol %
(
-
1
-2
7
isotonic) to 0.0079 min mol % (hypotonic) (Figure 1). With
the 13-atm dispersion, the value of k /K increased to 0.011 min
2
-
1
-
2
mol %
.
Related experiments that were carried out, in which only the
shift reagent was present in the external aqueous phase (i.e., LiCl
*
To whom correspondence should be addressed. E-mail: SLR0@
Lehigh.edu.
1
1254 J. AM. CHEM. SOC. 2002, 124, 11254-11255
9
10.1021/ja026895s CCC: $22.00 © 2002 American Chemical Society

C O MMU N I C A T I O N S
When (C16:1)PC liposomes containing 2 were placed under
isotonic and hypotonic (517 mosM, 13 atm) conditions, the value
+
+
of k
error (i.e., k
2
/K for Na /Li transport was the same within experimental
/K was 0.66 ( 0.06 min- mol % ). These results
1
-2
2
imply that the ability of 1 to recognize osmotically stressed
phospholipid bilayers has more to do with the length of the
transport-active species than changes in the packing density of the
membrane. At the same time, they indicate that the ability of an
ion conductor to recognize osmotically stressed phospholipid
bilayers depends, significantly, upon its structure.
Figure 2. Plot of specific internal volume (9, L/mol of phospholipid) and
3
-1
-2
1
0 k2/K (b, min mol % ) as a function of applied osmotic gradient for
2
00-nm vesicles made from (C16:1)PC; the dotted line represents a
hypothetical curve for vesicles exhibiting ideal osmotic behavior.
was absent) and 1.5 mol % of 1 was present in the liposomes,
+
showed negligible Na efflux; addition of LiCl to the dispersion
+
+
resulted in rapid Na /Li transport. Such a finding is significant
In preliminary studies, we have examined the activity of 1 for
for two reasons. First, it establishes that the presence of 1 does not
destroy the integrity of the bilayer; that is, the movement of Na /
Li across the membrane takes place by a transport process and
not via leakage through defects. Second, it lends strong support
+
+
Na /Li transport across liposomes of (C16:1)PC under isotonic
and hypotonic conditions when 1 is added, externally, to preformed
liposomes; that is, the ion conductor is introduced under “single-
sided” conditions. Qualitatively, the results that were obtained were
similar to those found under “double-sided” conditions, where the
ion conductor was present during liposome formation. Thus, the
+
+
3
+
for an antiport mechanism in which Na efflux is compensated by
+
Li influx.
To confirm the osmotic responsiveness of liposomes made from
C16:1 PC), we measured their changes in internal volume when
exposed to varying hypotonic solutions. Thus, using procedures
similar to those previously described, 5(6)-carboxyfluorescein (CF)
was encapsulated within large unilamellar vesicles (200 nm), and
its self-quenching efficiency (Q) plotted as a function of the internal
2
values of k /K under isotonic and hypotonic (517 mosM, 13 atm)
(
-1
-2
conditions were 0.0044 and 0.041 min mol % , respectively.
Efforts currently in progress are aimed at examining, in greater
detail, the relationships that exist between the structure of an ion
conductor and its osmotic stress-recognition behavior with a view
8
toward drug design. The results of these studies will be reported
3,4
CF concentration under isotonic conditions. Here, Q is defined
as a percentage such that Q(%) ) 100[1 - (I/I )], where I is the
in due course.
o
o
Acknowledgment. We are grateful to the National Science
total fluorescence intensity of the dispersion after complete release
by Triton X-100, and I is the fluorescence intensity of the entrapped
CF. Using this plot as a calibration curve, the changes in internal
volume (as indicated by changes in internal CF concentrations)
could then be determined under hypotonic conditions. A plot of
internal volume as a function of the initial osmotic gradient is shown
in Figure 2. Also shown in this figure is a hypothetical curve for
liposomes exhibiting ideal osmotic behavior. Similar to what has
been observed previously, the osmotic properties of such liposomes
Foundation (Grant CHE-9986704) for support of this research.
Supporting Information Available: Procedures for the synthesis
of 2 and ion transport measurements; plots of kobsd versus (mol % 2)
2
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(
1) Bandyopadhyay, P.; Janout, V.; Zhang, L.; Regen, S. L. J. Am. Chem.
Soc. 2001, 123, 7691.
2
were found to be nonideal. These results clearly show that a
(2) (a) Bummer, P. M.; Zografi, G. J. Pharm. Sci. 1988, 77, 1021. (b) Naka,
K.; Sadownik, A.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 2278.
considerable amount of osmotic pressure has been converted into
osmotic stress.
(
3) Melittin and certain wedge-shaped surfactants disrupt the integrity of
osmotically stressed liposomal membranes, releasing calcein and 5(6)-
carboxyfluorescein, more efficiently than similar membranes held under
+
+
The sensitivity of 1 toward Na /Li transport in osmotically
stressed bilayers is signficant. How much of this recognition is due
to a proper matching of the length of the transport-active species
with the thickness of the bilayer or to a decrease in the packing
density of the membrane is not apparent from these results. It was
of interest, therefore, to compare the stress-recognition behavior
of an ion conductor that shows only a modest dependency on bilayer
thickness. One such compound is the sterol-polyether conjugate,
2b,4
isotonic conditions. In contrast, liposomes that contain 1 do not release
+
+
Na
until Li is added, externally; i.e., 1 is not membrane-disrupting.
(
(
4) Benachir, T.; Lafleur, M. Biophys. J. 1996, 70, 831.
5) Pregel, M. J.; Jullien, L.; Lehn, J. M. Angew. Chem., Int. Ed. Engl. 1992,
3
1, 1637.
(
(
6) Otto, S.; Osifchin, M.; Regen, S. L. J. Am. Chem. Soc. 1999, 121, 7276.
7) A similar experiment that was carried out using 1 mol % of 1 and a 10-
atm osmotic gradient, with lower salt concentrations (the internal NaCl
and external LiCl concentrations were reduced from 450 and 208 mM to
2
50 and 49 mM, respectively) gave the same rate constant, within
experimental error ((10%). Thus, ionic strength is not a significant factor
2. Similar to that for 1, kobsd exhibits a second-order dependency
in these experiments.
(
8) Hypotonicity produces osmotic pressures of ca. 15-20 and 0.9-5 atm in
Gram positive and Gram negative bacteria, respectively. In contrast,
mammalian membranes are relatively stress-free: Csonka, L. N. Microbiol.
ReV. 1989, 53, 121.
on the mol % of 2 that is present (see Supporting Information).
The ion-transport activity of 2, however, shows a relatively modest
dependence on bilayer thickness; its activity in (C16:1)PC mem-
branes is ca. 20 times greater than in (C18:1)PC liposomes.
JA026895S
J. AM. CHEM. SOC.
9
VOL. 124, NO. 38, 2002 11255