Triggered Release from Lipid Bilayer Vesicles by an Artificial Transmembrane Signal Transduction System

Article abstract of DOI:10.1021/jacs.7b07747

The on-demand delivery of drug molecules from nanoscale carriers with spatiotemporal control is a key challenge in modern medicine. Here we show that lipid bilayer vesicles (liposomes) can be triggered to release an encapsulated molecular cargo in response to an external control signal by employing an artificial transmembrane signal transduction mechanism. A synthetic signal transducer embedded in the lipid bilayer membrane acts as a switchable catalyst, catalyzing the formation of surfactant molecules inside the vesicle in response to a change in external pH. The surfactant permeabilizes the lipid bilayer membrane to facilitate release of an encapsulated hydrophilic cargo. In the absence of the pH control signal, the catalyst is inactive, and the cargo remains encapsulated within the vesicle.

Full text of DOI:10.1021/jacs.7b07747

Article
pubs.acs.org/JACS
Triggered Release from Lipid Bilayer Vesicles by an Artificial
Transmembrane Signal Transduction System
Matthew J. Langton,† Lorel M. Scriven, Nicholas H. Williams,* and Christopher A. Hunter*,†
†
,‡
†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
‡Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom
*
S Supporting Information
ABSTRACT: The on-demand delivery of drug molecules from nanoscale
carriers with spatiotemporal control is a key challenge in modern medicine.
Here we show that lipid bilayer vesicles (liposomes) can be triggered to
release an encapsulated molecular cargo in response to an external control
signal by employing an artificial transmembrane signal transduction
mechanism. A synthetic signal transducer embedded in the lipid bilayer
membrane acts as a switchable catalyst, catalyzing the formation of
surfactant molecules inside the vesicle in response to a change in external
pH. The surfactant permeabilizes the lipid bilayer membrane to facilitate
release of an encapsulated hydrophilic cargo. In the absence of the pH
control signal, the catalyst is inactive, and the cargo remains encapsulated
within the vesicle.
1
9,20
INTRODUCTION
range of therapeutics,
and there are established clinical
■
2
1,22
applications in the delivery of anticancer agents.
In recent
Transmembrane signal transduction and signal amplification
are important features of numerous biological processes,
facilitating the communication of chemical information across
years, significant research effort has been applied to developing
responsive liposomes, where the release of a payload is
triggered in response to an external or environmental stimulus
1
lipid bilayer membranes. In Nature, signaling is achieved by
23
(
e.g., pH, enzymes, and temperature). Here we show that an
membrane-spanning proteins, such as G-protein-coupled
2
artificial signal transduction mechanism can be used to control
the formation of surfactants inside lipid bilayer vesicles,
triggering the release of an internal cargo in response to a
change in external pH.
receptors, that generate an intracellular signal (second
messenger) in response to binding of an extra-cellular signaling
molecule (first messenger). Second messengers trigger signal-
ing cascades that lead to the cellular response. Typically, such
cascades also amplify the signal by initiating enzyme-catalyzed
processes. Many synthetic supramolecular systems capable of
physically transporting chemical signals, such as ions, across
lipid bilayer membranes have been reported.
mimicking signal transduction and amplification processes that
occur without physical transfer of matter has proved a
considerable challenge for synthetic systems.
APPROACH
■
The addition of amphiphilic surfactants to lipid bilayer vesicles
leads to lysis by solubilization of the lipids.
sub-solubilizing concentrations, surfactants also have profound
effects on the properties of lipid bilayers, leading to curvature
strain, alkyl chain disordering, lateral expansion, and increased
permeability to hydrophilic solutes without destruction of the
vesicle. To exploit this effect, we employed an artificial
transmembrane signal transducer to initiate the production of
low concentrations of surfactant inside lipid bilayer vesicles
3
−9
However,
24−26
However, at
1
0−15
Recently,
we have shown that a new abiotic mechanismmembrane
translocationcan be used for signal transduction across lipid
bilayers in artificial systems (Figure 1a). The reversible
movement of a synthetic transducer molecule across a bilayer
can be controlled by chemical input signals on the outside of
the vesicle. The position of the transducer is coupled to catalyst
activation, so that an amplified output signal is generated on the
27
(
Figure 1b). The controlled generation of surfactant provides a
mechanism for the release of encapsulated cargo by
permeabilising the lipid bilayer (Figure 1c). While the synthesis
of surfactant molecules in bilayer systems has been explored in
the context of self-replicating fatty acid vesicles, in which the
auto)catalytic formation of fatty acids from precursors leads to
self-reproduction of the vesicles, the controlled production of
16,17
inside of the vesicle.
One outcome of biological transmembrane signal trans-
duction can be the activation of channels that control the influx
or efflux of ions, such as calcium, in response to extra-cellular
(
28
1
,18
signals.
We hypothesized that a transmembrane signaling
process could be used in a similar way to trigger release of
membrane-impermeable solutes from inside vesicles. Lipid
bilayer vesicles are attractive candidates for the delivery of a
Received: July 24, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b07747
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Journal of the American Chemical Society
Article
Figure 1. Triggered cargo release from vesicles using an artificial signal transduction mechanism. (a) Transmembrane signal transduction. The input
signal switches the external headgroup of a synthetic signal transducer embedded in the membrane from polar (blue) to apolar (purple), allowing it
to translocate through the membrane. Binding of a charged cofactor to the inner headgroup (red) activates the catalyst (green). (b) Catalytic
surfactant generation. The catalyst hydrolyzes a substrate (gray), generating a surfactant (yellow). (c) Cargo release. The surfactant enhances the
+
permeability of the membrane to polar solutes, facilitating cargo release (pink). (d) Molecular structures of protonated signal transducer 1·H (OFF
2
+
state) and the activated catalyst 1·Zn (ON state).
surfactants to release a cargo from lipid bilayer vesicles has not
been explored.
The triggered release process is shown schematically in
Figure 1. A high concentration of the water-soluble cargo
Cargo Release Experiments. Identification of a suitable
surfactant that could be generated using the signal transduction
system was addressed first. In the ON state of the transducer
+
(1·Zn ), the zinc-oxime complex catalyzes the hydrolysis of
(
pink) and a low concentration of the hydrophilic surfactant
ester substrates, so the catalyst could be used to generate a
hydrophobic carboxylic acid as the second messenger surfactant
product. We identified 2-naphthoic acid as a potential second
messenger (compound 2, Figure 2). This compound is a
precursor (gray) are encapsulated in vesicles that have a
synthetic signal transducer embedded in the membrane.
The first stage of the process is transduction of the chemical
signal across the lipid bilayer membrane by the synthetic
29
30
known hydrotrope and membrane-permeable. The surfac-
tant properties were assessed using calcein release experiments.
Calcein is a membrane-impermeable fluorophore (Figure 2)
that self-quenches at high concentrations. When calcein is
loaded inside vesicles at a concentration of 70 mM self-
quenching occurs. Any calcein efflux from the vesicles results in
fluorophore dilution and reduction in the extent of self-
quenching, which is detected by an increase in the calcein
emission. Calcein fluorescence can therefore be used as an
16,17
transducer (Figure 1a).
The transducer is shorter than the
width of the bilayer, so when the external recognition
headgroup is in a charged state, it preferentially sits in the
aqueous phase, and the transducer is located in the outer leaflet.
The input signal (the first messenger, orange) switches the
external headgroup to an apolar state, allowing the transducer
to translocate through the membrane. When the internal
headgroup binds a charged cofactor, it preferentially sits in the
aqueous phase, fixing the transducer in the inner leaflet. In the
second step (Figure 1b), the activated catalyst hydrolyses the
encapsulated substrate (gray) to generate the internal second
messenger, a surfactant (yellow). In the final stage, the
hydrophobic surfactant embeds in the lipid bilayer membrane,
rendering it permeable to the cargo molecules which diffuse out
of the vesicle (Figure 1c). In this work, we used the pH-
31
indicator for lipid vesicle leakage. The extent of calcein release
can be quantified by lysis of the vesicles with detergent (Triton
X-100), allowing the change in fluorescence emission intensity
to be calibrated relative to release of the entire contents of the
vesicles.
Vesicles containing a self-quenched calcein solution were
assembled by hydrating a dried mixture of 1,2-dioleoyl-sn-
glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glyc-
ero-3-phosphoethanolamine (DOPE) lipids in an aqueous
solution containing 70 mM calcein in HEPES buffer at pH 7.
1
6
controlled signal transducer 1 to initiate intravesicle
surfactant generation (Figure 1d). Transducer 1 features a
morpholine headgroup, which is protonated at neutral pH (1·
+
H ) and can be deprotonated by raising the pH on the outside
32
of the vesicle to initiate translocation. The pro-catalyst
headgroup is a neutral pyridine oxime, which becomes a
The suspension was extruded through a polycarbonate
membrane to afford vesicles of an average diameter of around
200 nm. The vesicles were separated from the external solution
using size exclusion chromatography and then suspended in a
non-buffered sodium chloride solution (pH 7). An aliquot of a
solution of 2 in methanol was added to the vesicle suspension,
and rapid release of calcein from the vesicles was observed
(black data, Figure 2b). In the absence of 2 (blue data) or upon
addition of methanol (gray data), the calcein remained
encapsulated, and the vesicles were stable until the detergent
+
catalyst for ester hydrolysis when it binds zinc (1·Zn ).
RESULTS AND DISCUSSION
■
The triggered release mechanism shown in Figure 1 involves
the cooperation of multiple functional components: (a)
transmembrane signal transduction, (b) catalytic surfactant
generation, and (c) cargo release. To address this complexity,
we investigated the behavior of each component individually,
prior to assembling the complete functional system.
33
was added. These experiments demonstrate that 2 has
B
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fluorescent. Cleavage of the ester reveals fluorescent pyranine 4
(
8-hydroxypyrene-1,3,6-trisulfonate), which enables convenient
monitoring of the reaction and quantification of the production
of surfactant 2.
Signal transducer 1 was synthesized as previously
1
6
described. The functional vesicles were assembled by
hydrating a dried mixture of lipids and transducer 1 in an
aqueous solution containing substrate 3, zinc chloride, and
HEPES buffer at pH 7. The suspension was extruded, the
vesicles were separated using size exclusion chromatography
and then suspended in sodium chloride solution to afford final
bulk concentrations of 2 mM lipids and 50 μM transducer. A
control system was prepared in the same way, but in the
absence of transducer 1. Incorporation of the transducer during
vesicle preparation results in a statistical distribution of
transducer 1 in the inner and outer leaflets of the membrane.
However, only the molecules in the outer leaflet are in the
correct orientation to interact with the external signal and to
catalyze reactions inside the vesicle. The remaining 50% of the
transducers that are in an inverted orientation are not sensitive
to changes in external pH and cannot catalyze reactions inside
the vesicle, so they are silent in the experiments.
Figure 4 shows a signal transduction experiment with
substrate 3. Sodium hydroxide was added to the external
vesicle solution to initiate translocation of 1 across the
membrane, and the fluorescence emission due to formation
of 4 was measured.
In the OFF state at pH 7, conversion of 3 to 4 was minimal
(
red data, <4% after 800 min) and comparable to the
background solvolysis rate in the absence of transducer 1
black data). At pH 7, the external headgroup of the transducer
(
is protonated, trapping it in the outer leaflet, such that the
pyridine oxime headgroup is embedded inside the membrane in
an inactive state. Addition of an aliquot of base to the
suspension instantaneously raised the external pH to 9
Figure 2. Calcein release assay. (a) Compound 2 (yellow) was added
externally to 200 nm DOPC/DOPE vesicles loaded internally with 70
mM calcein (pink). The vesicles (lipid concentration 0.5 mM)
contained 250 mM HEPES at pH 7 and were suspended in 400 mM
NaCl. (b) Calcein release measured by fluorescence emission at 540
nm (exciting at 470 nm). Blue data: background leakage of calcein.
Gray data: background leakage of calcein in the presence of 4 μL of
methanol. Black data: release of calcein triggered by addition of 2 in 4
μL of methanol (final concentration 50 μM). Calcein release was
calibrated to 100% release by lysis with Triton X-100.
(
determined using a pH-electrode) and led to a sharp increase
in fluorescence intensity due to formation of 4. At pH 9, the
morpholine headgroup is deprotonated, so transducer 1 can
translocate across the bilayer, generating the ON state in which
the zinc−pyridine oxime complex catalyzes the hydrolysis of 3
inside the vesicles (green data, Figure 4). These experiments
demonstrate that ester 3 is a suitable substrate for the signal
transduction system and that surfactant 2 can be generated in
response to an external pH signal.
suitable surfactant properties to act as the second messenger
illustrated in Figure 1.
The experiments also provide some insight into how
surfactant 2 generated by the in situ hydrolysis reaction affects
the integrity of the vesicle membranes. The other product of
the hydrolysis reaction 4 acts as a fluorescent sensor for local
Catalytic Surfactant Generation. Having identified
naphthoic acid 2 as a suitable second messenger for triggering
cargo release, we turned our attention to generating this
surfactant in situ from an ester precursor encapsulated inside
vesicles (Figure 3). The ester substrate for the hydrolysis
reaction, 3, was prepared in one step from 4 and 2-naphthoyl
chloride (see Supporting Information). Substrate 3 is water-
soluble, highly charged, membrane-impermeable, and non-
34
pH. In the experiments described above, vesicles were
prepared with an internal pH of 7, and in the OFF state, this
pH was maintained. However, in the ON state, the apparent
value of the internal pH increased with time, reaching a value of
35
around pH 8 after 60 min (Figure 5).
This observation can be rationalized in two ways. One
explanation is that 2-naphthoic acid 2 generated by the
catalyzed hydrolysis of 3 enhances the permeability of the
+
−
membrane with respect to H /OH . Since the external pH is 9
and the internal pH is 7, equilibration of the proton gradient
across the membrane would lead to an increase of pH on the
inside of the vesicles, where it is reported by encapsulated 4.
The alterative explanation is that 2 causes the membrane to
become permeable to 4 which diffuses out of the vesicles. In
this case, the apparent pH is a population-weighted average
Figure 3. Molecular structures of ester substrate 3 and hydrolysis
products, surfactant 2 and fluorophore 4.
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analyzed by re-purifying the vesicle suspension using size
exclusion chromatography. This process removes any small
molecules that are not encapsulated inside vesicles. After
purification, a significant reduction in the intensity of the
fluorescence emission arising from 4 was observed (Figure 6),
demonstrating that formation of surfactant 2 leads to leakage of
4
from the vesicles.
Figure 6. Fluorescence excitation spectrum (emission at 510 nm) of
vesicles at the end of a signal transduction experiment (ON state after
1
000 min, experimental conditions as in Figure 4). Spectra were
recorded before (black data) and after (blue data) SEC purification to
remove extra-vesicle material and normalized to account for dilution
during purification.
Transmembrane Signal Transduction-Triggered Re-
lease Experiments. Having established that the individual
components of the system function separately, calcein release
initiated by in situ generation of 2 by transmembrane signal
transduction was investigated.
Vesicles containing membrane-embedded transducer 1 and
encapsulating 70 mM calcein, 25 μM 3, and 250 μM ZnCl2
were prepared. The results of the triggered release experiments
are shown in Figure 7. Addition of hydroxide to switch the
transducer to the ON state led to rapid release of calcein from
the vesicles only when the ester substrate was also present
inside the vesicles (green data in Figure 7b). This shows that
the three stages of the process (signal transduction, catalytic
generation of surfactant, and cargo release) can cooperate in
the fully functional controlled release mechanism, facilitating
cargo release from inside vesicles in response to an extra-vesicle
signal. After 800 min, the amount of calcein released is 15 times
greater than the amount of hydroxide added to initiate
signaling. The observed signal amplification is due to catalysis
of production of the second messenger, which is itself a catalyst
for transport of the output signal across the lipid membrane.
Such catalytic cascade processes are one of the hallmarks of
signal amplification in biological systems.
Figure 4. (a) Surfactant generation inside vesicles controlled by
transmembrane signal transduction. Activation of transducer 1 by
extra-vesicle hydroxide leads to catalyzed hydrolysis of 3 (gray) inside
the vesicles to generate surfactant 2 (yellow), which is quantified by
measuring the fluorescence emission arising from formation of
fluorophore 4. (b) Time dependence of the normalized fluorescence
emission intensity at 510 nm (exciting at 415 nm) of vesicles in the
presence (red data) and absence (black data) of transducer 1, and
following NaOH addition (green data) to initiate signal transduction.
All experiments were conducted in 200 nm DOPC/DOPE vesicles (2
mM final lipid concentration) containing 250 μM ester 3, 250 μM
ZnCl , and 250 mM HEPES buffer at pH 7, and suspended in 250 mM
2
NaCl. The loading of transducer 1 was 2.5 mol%.
To determine whether cargo release occurs by complete lysis
of the vesicles or by permeabilization of intact vesicles, size
exclusion chromatography was used to re-isolate the vesicles at
the end of the experiment. Analysis of the purified vesicles by
dynamic light scattering showed that the size distribution of the
vesicles obtained after calcein release (ON state) was identical
to that of the precursors in the OFF state (see Supporting
Figure 5. Effect of transmembrane signal transduction on the apparent
local pH determined spectroscopically by ratiometric analysis of the
fluorescence emission from 4 (experimental conditions as in Figure 4).
Gray data: vesicles incubated at pH 7 (OFF state). Black data: vesicles
incubated at pH 7, then pH 9 (arrow indicates addition of hydroxide
to form the ON state).
1
Information). H NMR experiments show that the total
concentration of phospholipid present in vesicles was
36
unchanged in the re-isolated vesicles after calcein release.
Purified vesicles from the ON and OFF experiments were
separately dissolved in a mixture of chloroform and methanol
containing an internal standard (3-(trimethylsilyl)propionic-
value determined by the amount of 4 inside and outside of the
vesicles. To distinguish these two scenarios, the integrity of the
vesicles after the transmembrane signaling reaction was
2,2,3,3-d acid sodium salt). Integration of the signal due to the
4
D
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ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b07747.
Synthetic procedures, characterization, and additional
experimental details (PDF)
AUTHOR INFORMATION
Corresponding Authors
n.h.williams@sheffield.ac.uk
herchelsmith.orgchem@ch.cam.ac.uk
■
*
*
ORCID
Matthew J. Langton: 0000-0003-1555-3479
Nicholas H. Williams: 0000-0002-4457-4220
Christopher A. Hunter: 0000-0002-5182-1859
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the University of Cambridge Oppenheimer Research
Fund for an Early Career Research Fellowship (M.J.L.).
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