Cytomimetic modeling in which one phospholipid liposome chemically attacks another [3]

Full text of DOI:10.1021/ja000504x

6492
J. Am. Chem. Soc. 2000, 122, 6492-6493
Cytomimetic Modeling in Which One Phospholipid
Liposome Chemically Attacks Another
Fredric M. Menger* and Vladimir A. Azov
Department of Chemistry, Emory UniVersity
Atlanta, Georgia 30322
ReceiVed February 10, 2000
Widespread membrane/membrane reactivity in biology (e.g.,
in viral attack upon cells and in sperm penetration of ova)1 would
lead one to expect considerable attention directed toward simple
“cytomimetic”2 models of such processes. This has not, however,
yet occurred. For example, the kinetics of bond-forming reactions
between two populations of liposomes (hollow spheres comprised
of bilayer membrane shells) are seemingly unknown.3,4 Studies
have been mainly confined to the bimolecular kinetics of species
in solution reacting with a species adsorbed onto a liposome.5-8
Thus, dithionite (S2 O42-) dissolved in water reduces a liposome-
bound fluorescent dye; the ensuing loss of fluorescence provided
rates of the solution/liposome reaction.9 On a more biological
level, it has been shown that water-soluble enzymes (chymotrypsin
and acetylcholineesterase) catalyze the hydrolyses of liposomal
substrates at rates that depend on the ability of the substrates to
project themselves beyond the membrane surface.10 It is the
purpose of the present work to examine liposome/liposome
chemistry and to compare it with the corresponding solution/
solution, solution/liposome, and intra-liposome reactions. The
variations are all depicted in Figure 1.
Figure 1. Schematic representation of an inter-liposomal reaction
compared to (a) solution/solution, (b) solution/liposome, and (c) intra-
liposomal reactions.
Figure 2. Structures of nucleophile 1 and electrophiles 2a, 2b, and 2c
that were used for surface modification of the liposomes.
Liposomal reactions were made possible by endowing one set
of liposomes with nucleophilicity (via a hydroxamate) and another
set of liposomes with electrophilicity (via a p-nitrophenyl
ester) using the compounds in Figure 2.11
Liposomes were prepared by hydrating for about 5 min at room
temperature a cast film composed of 2 mg of phospholipid (1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) plus 10 mol %
of 1 or 2 (0.15-0.20 mg) in 1 mL of gently stirred buffer. The
resulting suspension was passed back and forth 19 times through
a polycarbonate membrane (100 nm pore diameter) with the aid
of a LiposoFast vesicle extruder.13 The liposomes had mean
hydrodynamic diameters of 120 ( 15 nm (measured routinely
with a Coulter N4 particle sizer before and after the kinetic runs).
A liposome preparation was diluted with buffer, mixed with a
water-soluble reagent or second liposome preparation, and assayed
at 400 nm and 25.0 °C for ester hydrolysis.
Note that addition of 1 or 2 (as an acetonitrile solution) to
preformed liposomes composed of pure phospholipid is an
unsatisfactory means of fabricating chemically reactive liposomes
because the additives precipitate in water before they have a
chance to enter the membrane. Owing to this water insolubility
of 1 and 2, no chemical reaction can occur between them in
aqueous buffer. This fact, plus the normal translucency of the
liposome preparations obtained from 1 or 2 and phospholipid,
establish that the reactants are membrane-bound.
Before describing liposome/liposome reactivity, it will be
helpful to discuss, by way of calibration, the rates of the simpler
systems in Figure 1. p-Nitrophenyl acetate (pNPA), at pH 9.0
and 25.0 °C in the absence of any nucleophile, hydrolyzes
“spontaneously” with an observed rate constant of only 2.0 ×
10-2 min-1 (half-life t1/2 ) 35 min). A mixture of pNPA (1.0 ×
10-4 M) and acetohydroxamate AH (1.0 × 10-3 M) under the
same conditions gave an observed rate constant kobs ) 2.0 min-1
(t1/2 ) 0.35 min). These values agree with literature data.14
1
2
Under the basic conditions of our experiments (pH 9.0), the acyl-
hydroxamate intermediate rapidly hydrolyzes to regenerate hy-
droxamate, thereby allowing for turnover and no net consumption
of nucleophile. The byproduct, p-nitrophenolate, has a strong
absorbance at 400 nm from which the rate of reaction can be
determined.
Nucleophilic and electrophilic entities were incorporated into
liposomal membranes via a cholesterol unit derivatized at its
3-position as shown in compounds 1 and 2a, 2b, 2c, respectively
(Figure 2). Electrophiles 2a, 2b, and 2c differ only in the length
of their hydrophilic spacers separating the ester functionality from
the cholesterol moiety. Cholesterol was selected as the liposomal
“anchor” owing to its known affinity for bilayer membranes.12
(1) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D.
Molecular Biology of the Cell; Garland Publishing: New York and London,
1989.
(2) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. Engl. 1995,
34, 2091-2106.
(3) Constable, E. C.; Meier, W.; Nardin, C.; Mundwiler, S. Chem. Commun.
1999, 1483-1484.
(4) Marchi-Artzner, V.; Lehn, J.-M.; Kunitake, T. Langmuir 1998, 14,
6470-6478.
(5) Lasic, D. D. Liposomes: from Physiscs to Application; Elsevier:
Amsterdam, 1993.
(6) Fuhrhop, J.-H.; Mathieu, J. Angew. Chem., Int. Ed. Engl. 1984, 23,
100-113.
(7) Mizutani, T.; Whitten, D. G. J. Am. Chem. Soc. 1985, 107, 3621-
3625.
(8) Kunitake, T.; Ihara, H.; Okahata, Y. J. Am. Chem. Soc. 1983, 105,
6070-6078.
(9) Moss, R. A.; Bhattacharya, S. J. Am. Chem. Soc. 1995, 117, 8688-
(12) Gabdoulline, R. R.; Vanderkooi, G.; Zheng, C. J. Phys. Chem. 1996,
100, 15942-15946.
(13) MacDonald, R. C.; MacDonald, R. I.; Mences, B. P. M.; Takeshita,
N. K.; Subbaras, N. K.; Hu, L. R. Biochim. Biophys. Acta 1991, 1061, 297-
303.
8689.
(10) Menger, F. M.; Nelson, K. H.; Guo, Y. Chem. Commun. 1998, 2001-
2002.
(11) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1960, 82, 1778-1786.
10.1021/ja000504x CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/21/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6493
Table 1. Summary of Rates for Reactions in Figure 1a
103[hydrox- 104[ester],
amate], mol/Lb mol/Lc,f
kobs
,
t1/2,
min
d
pH
min-1
pNPA, buffer
AH + pNPA
2aves, buffer
AH + 2aves
AH + 2aves
1ves + pNPA
1ves + 2aves
(1 + 2a)ves
1.0
1.0
1.0
1.0
9.0 2.0 × 10-2
35
1.0
9.0 2.0
0.35
180
7.7
38
7
120
9.0 3.8 × 10-3
9.0 9.0 × 10-2
9.0 1.8 × 10-2
9.0 9.8 × 10-2
9.0 5.8 × 10-3
8.0e 2.1 (stage 1)
0.12 (stage 2)
1.0
0.10
0.10
0.10
0.10
1.0
0.50
0.50
0.50
0.3
6
a All measurements were performed at 25 ( 0.3 °C in 0.1 M
carbonate or 0.1 M phosphate buffer. b Concentration of AH or
liposome-bound 1. c Concentration of pNPA or liposome-bound ester
2a. d Rate constants are reproducible to (10%. e Reactions were too
fast to follow at pH 9.0. f 2a, 2b, and 2c all react at similar rates.
Figure 3. Absorbance at 400 nm for the biphasic 1 + 2a intra-liposomal
reaction (pH 8.0, 25.0 °C). The horizontal dashed line represents an
approximate “infinity” for the first stage involving reaction within the
outer leaflet of the membrane bilayer.
When ester 2a is bound to the liposomes, it hydrolyzes more
slowly under the standard conditions than does “free” pNPA.
Thus, “spontaneous” hydrolysis of liposomal 2a in the absence
of nucleophile has a t1/2 of 180 min instead of the 35 min for
aqueous pNPA. The rate decrease also manifests itself in the
bimolecular reaction between liposomal ester (1.0 × 10-4 M) and
solution-phase AH (1.0 × 10-3 M) where t1/2 increases from 0.35
min for the solution/solution reaction to 7.7 min for the corre-
sponding solution/liposome reaction. Clearly, accessibility factors
at the membrane surface help protect the ester from nucleophilic
attack by external AH. This could likely occur by partial burying
of the ester group via looping of the spacer separating it from
the embedded cholesterol moiety.15,16
It was also of interest to interchange reactants in the solution/
liposome reaction by placing hydroxamate 1 in the liposome and
the ester (pNPA) in the external water. We needed here to reduce
the hydroxamate concentration 10-fold to 1.0 × 10-4 M in order
not to overload the liposome with more than 10 mol % of guest
nucleophile. In any event, these liposomes reacted with 0.5 ×
10-4 M pNPA (a cleanly first-order process characteristic of
turnover) at a rate of 9.8 × 10-2 min-1 (t1/2 ) 7 min). If this rate
is elevated 10-fold to adjust for the lower hydroxamate concentra-
tion relative to that of the solution/solution reaction, then one
sees that the liposomal rate is only a factor of 2 less than that of
the nonvesicular AH + pNPA reaction. In other words, liposomal
nucleophile and “free” nucleophile have similar reactivities (in
contrast to the situation with liposomal and “free” ester). The
difference between hydroxamate and ester must be that the former
is anionic and, therefore, the hydroxamate of liposomal 1 tends
to remain fully exposed to the bulk water.
We are now in position to discuss the liposome/liposome
reaction carried out with 1.0 × 10-4 M 1 in one set of liposomes
and 0.5 × 10-4 M 2a in another. The observed rate constant of
5.8 × 10-3 min-1 (t1/2)120 min) is similar to, but definitely faster
than, “spontaneous” buffer hydrolysis of liposomal 2a (which,
as mentioned, has a t1/2)180 min). Multiple repeat runs ensure
that the liposome/liposome chemical reaction is real and, to our
knowledge, the first instance of its kind. At this point it is not
possible to specify the number of hydrolytic events per liposome/
liposome collision.
Dynamic light scattering experiments upon completion of the
liposome/liposome reactions showed no significant particles larger
than the original 120 nm liposomes. This is consistent with
turnover, i.e. two liposomes will chemically couple to each other
after which the acyl intermediate hydrolyzes, the liposomes
become quickly independent once again, and the hydroxamate
concentration remains constant.
By far the fastest membrane reaction occurred when the
nucleophile and electrophile (at comparable concentrations) were
contained within one and the same bilayer. In fact, the intra-
liposomal reaction was so fast that we had to make two adjust-
ments to our procedure: (a) Since hydration and extrusion of a
phospholipid film containing 1 and 2 gave immediate hydrolysis
under basic conditions, the film hydration was carried out at pH
∼3. Extrusion of the acidic suspension produced the liposomes.
(b) Reactions were then initiated by rapidly bringing the pH up
to 8.0 (not the usual 9.0). A one-unit lowering of the pH slowed
the reaction by reducing roughly 8-fold the percent of hydrox-
amate in the hydroxamic acid/hydroxamate mixture (pKa ) 9.4).17
A plot of p-nitrophenolate absorbance at 400 nm vs time shows
a decidedly biphasic behavior (Figure 3) when 1 (1.0 × 10-4 M)
and 2a (0.5 × 10-4 M) share a common membrane. This is most
simply interpreted as an initial fast intra-liposomal reaction (t1/2
) 0.3 min) between 1 and 2a within the outer leaflet of the
phospholipid bilayer. As the conjugate acid form of 1 in the inner
leaflet is converted into hydroxamate via inward diffusion of
hydroxide through the bilayer, or as ester 2a flip-flops from the
inner to outer leaflets, the remaining half of the reaction can
proceed. This second and slower component of the biphasic
process has a half-life of about 6 min. Note that the chemistry is
proceeding here at a pH of only 8.0 and 0.1 mM hydroxamate.
Adjusting the rate constants upward (to correct for the lower pH
and for the lower hydroxamate concentration relative to that of
the AH/pNPA reaction) leads to the conclusion that the intra-
liposomal reaction is roughly 1-2 orders of magnitude faster than
even the solution/solution reaction. Confinement of the bimo-
lecular reaction to the membrane obviously has a highly positive
effect upon the kinetics. Indeed, among the membrane reactions
only the intra-liposomal mode is sufficiently fast to allow detection
of accompanying diffusion-controlled processes.
In summary, we have determined that several modes of
liposomal reactivity under comparable conditions have rates of
decreasing magnitude according to the following: intra-liposome
> solution/solution > solution/liposome > inter-liposome. It
appears as if reactivity of a membrane surface depends markedly
upon the affinity of the relevant functionalities for the aqueous
medium. In our case, one functionality (the hydroxamate) is fully
exposed, whereas the other (the ester) is not, a situation that leads
to a slow but observable liposome/liposome reaction.
Acknowledgment. This work was supported by the National Institutes
of Health.
Supporting Information Available: Experimental details, charac-
terization of compounds, and additional kinetic data for 2b and 2c (PDF).
This material is available free of charge via the Internet at http://pubs.acs.org.
(14) Dessolin, M.; Laloi-Diard, M.; Vilkas, M. Bull. Soc. Chim. Fr. 1970,
2573-2580.
JA000504X
(15) Menger, F. M.; Richardson, S. D.; Wood, M. G., Jr.; Sherrod, M. J.
Langmuir 1989, 5, 833-838.
(16) Frisch, B.; Boeckler, C.; Schuber, F. Bioconj. Chem. 1996, 7, 180-
(17) Wise, W. M.; Brandt, W. W. J. Am. Chem. Soc. 1955, 77, 1058-
1059.
186.