Synthesis and Biophysical Characterization of an Odd-Numbered 1,3-Diamidophospholipid

Article abstract of DOI:10.1021/acs.langmuir.7b04227

Nanomedicine suffers from low drug delivery efficiencies. Mechanoresponsive vesicles could provide an alternative way to release active compounds triggered by the basic physics of the human body. 1,3-Diamidophospholipids with C16 tails proved to be an effective building block for mechanoresponsive vesicles, but their low main phase transition temperature prevents an effective application in humans. As the main phase transition temperature of a membrane depends on the fatty acyl chain length, we synthesized a C17 homologue of a 1,3-diamidophospholipid: Rad-PC-Rad. The elevated main phase transition temperature of Rad-PC-Rad allows mechanoresponsive drug delivery at body temperature. Herein, we report the biophysical properties of Rad-PC-Rad monolayer and bilayer membranes. Rad-PC-Rad is an ideal candidate for advancing the concept of physically triggered drug release.

Full text of DOI:10.1021/acs.langmuir.7b04227

Article
pubs.acs.org/Langmuir
Cite This: Langmuir XXXX, XXX, XXX−XXX
Synthesis and Biophysical Characterization of an Odd-Numbered 1,3-
Diamidophospholipid
Frederik Neuhaus,†,‡ Dennis Mueller,† Radu Tanasescu,† Sandor Balog,§ Takashi Ishikawa,∥
Gerald Brezesinski,⊥ and Andreas Zumbuehl*,†,‡
†Department of Chemistry, University of Fribourg, Chemin du Musee
́
9, 1700 Fribourg, Switzerland
‡National Centre of Competence in Research in Chemical Biology, Quai Ernest Ansermet 30, 1211 Geneva, Switzerland
§Adolphe Merkle Institute, University of Fribourg, Chemin du Verdiers 4, 1700 Fribourg, Switzerland
∥Paul Scherrer Institute (PSI), OFLB/010, 5232 Villigen PSI, Switzerland
⊥Max Planck Institute of Colloids and Interfaces, Research Campus Potsdam-Golm, 14476 Potsdam, Germany
S
* Supporting Information
ABSTRACT: Nanomedicine suffers from low drug delivery efficiencies.
Mechanoresponsive vesicles could provide an alternative way to release active
compounds triggered by the basic physics of the human body. 1,3-
Diamidophospholipids with C16 tails proved to be an effective building block
for mechanoresponsive vesicles, but their low main phase transition temperature
prevents an effective application in humans. As the main phase transition
temperature of a membrane depends on the fatty acyl chain length, we
synthesized a C17 homologue of a 1,3-diamidophospholipid: Rad-PC-Rad. The
elevated main phase transition temperature of Rad-PC-Rad allows mechanores-
ponsive drug delivery at body temperature. Herein, we report the biophysical
properties of Rad-PC-Rad monolayer and bilayer membranes. Rad-PC-Rad is an
ideal candidate for advancing the concept of physically triggered drug release.
Although being self-assembled from the artificial phospho-
lipid Pad-PC-Pad (2),3 the vesicles were found to be
surprisingly nontoxic and were not activating the complement
system in in vivo experiments.13,14 Therefore, Pad-PC-Pad (2)
vesicles might be ideal candidates for mechanoresponsive
vesicular drug delivery. However, the main phase transition of
Pad-PC-Pad (2) lies at Tm = 37 °C, and heating the vesicles to
this temperature will lead to the immediate full release of any
vesicle-entrapped cargo because of the leakiness of a liquid-
crystalline membrane10 and the leakiness of a lipid membrane
close to the chain-melting regime.15 It was therefore necessary
to increase the Tm of the membrane while maintaining the
mechanosensitivity. A straightforward solution is to formulate
vesicles from the C18 analogue16 of Pad-PC-Pad (2), termed
Sad-PC-Sad (3). Vesicles of Sad-PC-Sad (3) showed a Tm = 52
°C, but unfortunately, the mechanoresponsiveness of the
vesicles at room temperature was lost.10 Therefore, we have
now synthesized and characterized the C17 analogue of Pad-
PC-Pad (2). The correct term for a C17 fatty acid is margaric
acid and its abbreviation, M, is more typically associated with
myristic acid.17 This is the reason why we have chosen a letter
INTRODUCTION
■
The emerging field of mechanoresponsive drug delivery seeks
to harness changes in physics between the healthy and diseased
tissues as triggers for targeting drugs to the place where they are
needed.1−3 One interesting trigger is the shear force in blood
vessels: following Murray’s law, this force is balanced below 1.5
Pa throughout the entire vascular system to minimize the work
needed to transport blood in the body.4,5 At the site of a
stenosis, the shear force is at least ten times higher6,7 and this
change in force might be used to activate drug release from
either nanoparticle aggregates8 or lenticular vesicles.3
Compared to the natural phospholipid 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC, 1 see Figure 1), the 1,3-
diamidophospholipid 1,3-palmitoylamido-1,3-deoxy-sn-glycero-
2-phosphatidylcholine (Pad-PC-Pad, 2) features two major
differences: (i) the fatty acyl esters are replaced with amide
moieties capable of forming intermolecular H-bonding net-
works.9,10 (ii) The non-natural 1,3-arrangement is spacing the
tails apart, leading to the bilayer membrane interdigitation.10,11
The combination of these two effects leads to constrictions on
the vesicle geometry, and lenticular or d-form shaped vesicles
are formed.12 These vesicles are tight when left untouched on
the bench but release their cargo when they are mechanically
stimulated, for example, by shaking. In other words, these
vesicles are mechanoresponsive.
Received: December 13, 2017
Revised: February 16, 2018
Published: February 18, 2018
© XXXX American Chemical Society
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yield headgroup-protected phosphoramidate 7. The final
diamidophospholipid Rad-PC-Rad (4) was reached after acidic
headgroup deprotection and quaternization with dimethyl
sulfate. The overall yield after five steps was 37% starting
from the headgroup-protected dichloropropyl phosphorami-
date 5. The chemically pure diamidophospholipid Rad-PC-Rad
(4) was then characterized on a molecular level, as monolayers
at the air−water interface [two-dimensional (2D) model] and
as bulk systems (three-dimensional model).
Surface Pressure/Area per Molecule Isotherms. The
surface pressure/area per molecule isotherm measurements
were carried out on a Langmuir−Pockels trough, equipped with
a Wilhelmy paper balance (accuracy 0.2 mN/m), at the air−
water interface. The isotherms were measured at different
subphase temperatures between 5 and 25 °C (see Figure 2).
Figure 1. Schematic depiction of the natural phospholipid DPPC and
the 1,3 diamidophospholipids with C16 (Pad-PC-Pad), C17 (Rad-PC-
Rad), or C18 (Sad-PC-Sad) fatty acyl chains.
between P(almitoyl) and S(tearoyl) and named the molecule
described in this paper Rad-PC-Rad (4).
In this publication, we discuss the synthesis and the
characterization of Rad-PC-Rad (4) monolayers and bilayers
and demonstrate that Rad-PC-Rad (4) vesicles are ideal
candidates for further research into mechanoresponsive drug
delivery.
Figure 2. : Surface pressure/area per molecule isotherms of a Rad-PC-
Rad (4) monolayer at the air−water interface on a Langmuir−Pockels
trough with an aqueous subphase at different temperatures. The
surface pressure was recorded via a Wilhelmy paper balance (accuracy
0.2 mN/m).
RESULTS AND DISCUSSION
■
Synthesis. The 1,3-diamidophospholipid Rad-PC-Rad (4)
has been synthesized expanding from our previously reported
synthetic route (see Scheme 1 and Supporting Information).3
The substitution of phosphorus oxytrichloride with 1,3-
dichloropropanol led to a phosphorochloridate. The second
substitution with Boc-protected ethanolamine led to the
reasonably stable phosphoramidate 5. This intermediate was
transformed into a diazide, followed by a reduction to the
diamine. The heptadecanoyl chains were accessed via
heptadecanoic acid chloride (6) prepared from heptadecanoic
acid with thionyl chloride and were reacted with the diamine to
Rad-PC-Rad (4) monolayers showed a typical liquid-
expanded (LE) to liquid-condensed (LC) phase behavior.
Above 10 °C, the plateau of an expanded/condensed phase
coexistence was observed. The appearance of the plateau is
accompanied by a shift of the initial lift-off area per molecule to
higher values. The plateau represents the first-order phase
transition from a LE to LC phase. As observed for several
diamidophospholipids, the plateau is not fully horizontal,
especially it becomes steeper as the temperature increases.9
The monolayers themselves are stable over the measurable
Scheme 1. Synthesis of Rad-PC-Rad (4) Starting from Dichloridate 5, Adding the Odd-Numbered Fatty Acid Chain
Heptadecanoic Acid Chloride (6) Followed by the Deprotection and Quaternization of Intermediate 7
B
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Figure 3. Temperature dependence of the main phase transition pressure πt (left) and temperature dependence of the entropy change (ΔS) (right)
at the main phase transition of Rad-PC-Rad (4) monolayers spread at the air−water interface.
Table 1. Summarized Data from GIXD Measurements of Rad-PC-Rad (4) Monolayers at Different Temperatures and Surface
a
Pressures
T/°C
π/mN/m
30 QXY
degen.
non-degen.
a
b
c
α/deg
β/deg
γ/deg
dist
τ/°
Axy/Å2
A0/Å2
10
1.34
0.88
1.36
0.82
1.34
0.81
1.36
0.79
1.37
0.77
1.38
0.76
1.43
0.63
1.37
0.64
1.38
0.64
1.45
0.00
1.47
0.00
1.47
0.00
1.47
0.00
1.47
0.00
1.47
0.00
1.48
0.00
1.47
0.00
1.47
0.00
5.57
5.15
5.15
114.5
122.8
122.8
0.108
NN
38.0
NN
35.7
NN
35.8
NN
34.7
NN
33.9
NN
32.9
NN
27.1
NN
29.2
NN
28.8
NN
24.1
19.0
QZ
15
20
30
14
18
22
26
36
26
34
QXY
QZ
5.51
5.61
5.50
5.47
5.36
5.13
5.46
5.41
5.09
5.11
5.08
5.06
5.04
4.95
5.07
5.05
5.09
5.11
5.08
5.06
5.04
4.95
5.07
5.05
114.4
113.4
114.4
114.6
115.7
117.7
114.8
115.2
122.8
123.3
122.8
122.7
122.1
121.1
122.6
122.4
122.8
123.3
122.8
122.7
122.1
121.1
122.6
122.4
0.109
NN
23.6
24.0
23.5
23.3
22.9
21.7
23.3
23.1
19.1
19.4
19.3
19.3
19.2
19.3
20.3
20.2
QXY
QZ
0.127
NN
QXY
QZ
0.109
NN
QXY
QZ
0.105
NN
QXY
QZ
0.084
NN
QXY
QZ
0.046
NN
30
QXY
QZ
0.101
NN
QXY
QZ
0.094
NN
a
Axy: in-plane area per chain and A0: cross-sectional area per chain with A0 = Axy cos τ.
Figure 4. GIXD heatmaps of the diffraction intensities as a function of the in-plane QXY and out-of-plane QZ components of the scattering vector Q
for Rad-PC-Rad (4) at 20 °C and different surface pressures π (from left to right: 14, 18, 22, 26, and 36 mN/m).
range of surface pressures up to an instrument-based cutoff at
51 mN/m. From the onset of the plateau, it is possible to
determine the temperature-dependent main transition pressure
πt. For Rad-PC-Rad (4), this dependence can be described by a
linear function (see Figure 3).18 The experimental data only
deviate from this linear function in close vicinity of the
minimum transition temperature T0 below which the transition
into a condensed phase directly starts from the gas-analogue
state (resublimation process). For Rad-PC-Rad (4), this
temperature is 11 °C. The temperature can be compared to
the lift-off temperatures of the main phase transition of
homologous 1,3-diamidophospholipids, showing a linear trend
of T0 versus the chain length (see Figure S14) because T0 of the
C16 homologue (2) is 1.3 °C and that of the C18 homologue
(3) is 18 °C.9
From the area per molecule at the onset of the first-order
phase transition (A0) at πt and the area at the end of the
expanded−condensed phase coexistence (Ac), it is possible to
calculate the enthalpy change (ΔH) of the phase transition
using a modified 2D Clausius−Clapeyron equation.
C
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ΔH = (Ac − A0)T
Article
dπt
dT
below) (see Figures S10 and S11). In the previous work, we
studied this in depth and depicted interleaflet chain
interdigitation and hydrogen-bond networks as the main
driving forces for this behavior.10,26 The ratio between facetted
and spherical vesicles calculated from Figure S10 is 21:4. The
still occurring spherical shape might be due to small impurities
in the prepared lipid membranes, as already small amounts of
interface-active compounds can significantly influence the
membrane shape and behavior.27
With this, the entropy change dependency is represented by
ΔS = ΔH/T which is shown in Figure 3. The extrapolation of
ΔS to zero gives the critical temperature of Rad-PC-Rad (4)
monolayers of 33 °C. Above this temperature, no compression
of the monolayer to a condensed state is possible. Compared to
the critical temperatures of Pad-PC-Pad (2) (27 °C) and Sad-
PC-Sad (3) (41 °C),9 Rad-PC-Rad (4) fits well into the trend
of an increasing critical temperature with increasing chain
length.
The interdigitation of the leaflets in a membrane can be
directly measured from lateral cuts through the membrane in
the cryo-TEM image (see Figure 5). To collect statistically
Grazing Incidence-Angle X-ray Diffraction. Grazing
incidence-angle X-ray diffraction (GIXD) experiments provide
insights into the lattice structure of condensed monolayers on
the Ångstrom scale.19−23 The in-plane component of the
scattering vector (QXY) and out-of-plane component (QZ)
maxima of different measurements are presented in Table 1,
and exemplary heatmaps are shown in Figure 4 (for the
remaining heatmaps, see Supporting Information). For Rad-
PC-Rad (4), two Bragg Peaks have been observed for every
measured temperature/surface pressure combination. The
signal at higher QZ is a degenerated signal composed of two
equal signals. The Rad-PC-Rad (4) monolayers adopt an
orthorhombic lattice structure described by a distorted
rectangular unit cell, with a distortion in the NN (nearest
neighbor) direction and a fairly high tilt angle t (between 27°
and 36°) of the lipid tails tilted to the same NN direction. At 20
°C, the chain cross-sectional area (A0) is around 19.3 Å2, and
therefore only marginally larger than that observed at lower
temperatures (19.0 Å2 at 10 °C and 19.1 Å2 at 15 °C). This is a
typical value for chains in a herringbone packing mode. This
packing mode is supported by the distortion value obtained by
extrapolation of the distortion versus sin2(t) (see Figure S15).15
d0 amounts to −7.75 × 10−2 and is clearly different from zero
which would be expected if the tilt is the only source for lattice
distortion. In the present case, the herringbone packing is an
additional source and might be caused by a hydrogen-bonding
network between the headgroups.
With increasing surface pressure, the tilt angle t decreases to
0.41° per mN/m (see Figure S16). Assuming a constant cross-
sectional area in the condensed phase, the tilting phase
transition pressure can be determined from a plot of 1/cos(t)
versus the lateral pressure. Extrapolation to 1/cos(t) = 1 yields
a tilting pressure of 62.3 mN/m (see Figure S16). Such a phase
transition would usually be characterized by a kink in the
surface pressure/area isotherm as expected for a second-order
phase transition.19 However, the expected transition surface
pressure is too high to be observed with the used setup. The
tilting phase transition pressure can be compared to those of
other lipids, such as the C18-analogous diamidophospholipid
Sad-PC-Sad (3). A trend is visible, with longer fatty acyl chains
leading to higher tilting phase transition pressures.9 At 30 °C,
the cross-sectional area of Rad-PC-Rad (4) 20.2 Å2 is in the
expected range for rotator phases (for full data, see Table 1 as
well as Figure 3 and Supporting Information).9,24
Figure 5. Cryogenic transmission electron micrograph of Rad-PC-Rad
(4) vesicles and an inset of facetted vesicles. Both scale bars have a
length of 200 nm (for additional cryo-TEM images, see Supporting
Information).
relevant data, we measured 509 membrane cuts from Figure
S10 (see Figure S12) with a mean membrane thickness of 3.20
0.02 nm (N = 509). With two times 0.7 nm for the
headgroups, the hydrophobic layer is 1.8 nm thick.12 An all-
trans alkyl chain of 16 C-atoms has a length of 2.05 nm.28 Thus,
the alkyl chains in a noninterdigitated bilayer would have a tilt
angle of 64°, which is rather unlikely. Therefore, a full bilayer
interdigitation can be assumed.
Differential Scanning Calorimetry. Interdigitation of the
membrane leaflets also influences the membrane main-phase
transition temperature.29 In differential scanning calorimetry
experiments from Rad-PC-Rad (4) containing LUVs with
heating and cooling rates of 0.5 K/min, a main phase transition
temperature of 44.7 °C with a change in enthalpy of 24.16 kJ/
mol (see Figure S9) is observed. The main phase transition
temperature is lower compared to a natural 1,2-diester
phospholipid with margaric acid chains, which is 48.6 °C.17
Also, the value for the bilayer main phase transition
temperature is 11.5 K higher than the calculated critical
temperature from the monolayer experiments. This corrobo-
rates that an additional attractive force needs to be overcome in
bilayers which is not present in monolayers, most probably
stemming from interdigitation.
As the main phase transition temperature of Rad-PC-Rad (4)
containing vesicles is well above the human body temperature,
these mechanosensitive liposomes are of interest for targeted
drug release as described in the literature.3,30 To investigate the
potential of the liposomes in this regard, release experiments
were carried out.
Cryo-Transmission Electron Microscopy. All cryo-trans-
mission electron microscopy (cryo-TEM) images in this paper
were measured according to a previously described protocol.25
The cryo-TEM images recorded from large unilamellar vesicles
(LUVs) entirely made of Rad-PC-Rad (4) show a significant
number of strongly facetted shapes below the main phase
transition temperature of the membranes (Tm ≈ 45 °C, see
Release Experiments. Previously, we reported the 5(6)-
carboxyfluorescein release from Pad-PC-Pad (2) containing
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Figure 6. Release experiments using 5(6)-carboxyfluorescein-loaded Rad-PC-Rad (4) LUVs obtained via extrusion through a 100 nm track-edged
filter membrane (LUVET100). Triggered release was induced by shaking the samples for a specific amount of time on a vortex shaker. Pad-PC-Pad
(2) at 37 °C showed an immediate full release of its cargo without shaking and was therefore only measured once.
vesicles. As Pad-PC-Pad (2) shows a strong spontaneous
release already at temperatures slightly above its main phase
transition temperature (Tm = 37 °C),3 the use of this lipid at
the average human body temperature of 37 °C31 is not ideal.
Increasing the Tm promises improved release properties. With
the longer chain length compared to Pad-PC-Pad (2), Rad-PC-
Rad (4) has a higher main phase transition temperature of 44.7
°C. This leads to a lower leakage of the vesicles at 22 °C (room
temperature) as well as 37 °C (average human body
temperature) (see Figure 6).
Each sample was treated with Triton X-100 to induce a full
release of 5(6)-carboxyfluorescein.3 At a temperature of 22 °C,
Rad-PC-Rad (4) vesicles show a spontaneous release of 9%
5(6)-carboxyfluorescein after 7 days (see Figure S13), with a
good mechanically induced release of 24% after 60 s of
vortexing. Pad-PC-Pad (2) has a mechanically induced release
of 44% after 60 s of vortexing, the release depends on the
duration of vortexing with an immediate burst release and a
slower continuous release afterward. It shows a spontaneous
release of 15% after 7 days. DPPC (1) shows a mechanically
induced release of 1% after 60 s vortexing and a spontaneous
release after 7 days of 5%. At 37 °C, the mechanically induced
release of Rad-PC-Rad (4) after 60 s of vortexing is 34%,
whereas Pad-PC-Pad (2) shows 100% release without vortexing
and DPPC (1) shows 18% release after 60 s of vortexing. These
release experiments show the potential of Rad-PC-Rad (4)
containing vesicles for targeted mechanoresponsive drug release
at body temperature.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.lang-
muir.7b04227.
1
Synthesis, H NMR, 13C NMR of heptadecanoyl acid
1
chloride (6); synthesis, H NMR, 13C NMR, and 31P
NMR of tert-butyl (2-((amino((1,3-diheptadecan-ami-
dopropan-2-yl)oxy)phosphoryl)oxy)ethyl) and 1,3-di-
heptadecanamidopropan-2-yl (2-(trimethylammonio)-
ethyl) phosphate (Rad-PC-Rad, 4); differential scanning
calorimetry; vesicle preparation; cryo-TEM images;
release experiments; film balance measurements; and
GIXD measurements (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: andreas.zumbuehl@unifr.ch.
ORCID
Frederik Neuhaus: 0000-0003-2749-7208
Sandor Balog: 0000-0002-4847-9845
Andreas Zumbuehl: 0000-0002-8003-4432
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Parts of this research were carried out at PETRA III at DESY, a
member of the Helmholtz Association (HGF). The authors
thank Dr. Uta Ruett and Dr. Florian Bertram for assistance in
using beamline P08 and the Paul Scherrer Institut (PSI)
Electron Microscopy (EM) facility for technical help. This work
has been supported by the University of Fribourg and the Swiss
National Science Foundation through a stipend professorship
to A.Z. and through the National Centers of Competence in
Research (NCCR) in Chemical Biology and Bio-inspired
Materials.
CONCLUSIONS
■
In conclusion, we reported the synthesis of a symmetrical 1,3-
diamidophospholipid with C17 fatty acyl chains. The properties
of the Rad-PC-Rad monolayers more closely resemble those of
its C16 analogue compared to the C18 homologue. The bilayer
membrane is interdigitated, and the vesicles are mildly faceted.
The main phase transition temperature of 44.7 °C allows
mechanoresponsive fluorescent cargo release at 37 °C, whereas
the system remains stable at room temperature. Overall, Rad-
PC-Rad is a promising system for advancing the concept of
mechanoresponsive drug delivery. At temperatures of 20 °C
and below, the molecules exhibit a herringbone packing mode
in monolayers most probably caused by a hydrogen-bonding
network between the headgroups. Surprisingly, this tight
packing mode cannot be found at slightly higher temperatures
(30 °C) at which the cross-sectional area (20.2 Å2) indicates a
rotator phase.
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DOI: 10.1021/acs.langmuir.7b04227
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