Phosphate bioisostere containing amphiphiles: A novel class of squaramide-based lipids

Article abstract of DOI:10.1039/c6cc04089f

We describe a novel class of amphiphiles with squaramide moiety as a phosphate bioisostere. Most synthesized squaramide-based amphiphiles have the favorable physicochemical properties of lipids, such as: formation of stable liposomes or giant unilamellar vesicles in aqueous solution, high phase-transition temperature, low vesicle leakage and phospholipase resistance properties.

Full text of DOI:10.1039/c6cc04089f

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Phosphate bioisostere containing amphiphiles: a novel class of
squaramide-based Lipids
Abhishek Saha,‡ Subhankar Panda,‡ Saurav Paul,‡ and Debasis Manna*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We describe a novel class of amphiphiles with squaramide moiety
Although, the pKa values of squaramide moiety have not
as phosphate bioisostere. Most of these synthesized squaramide- been well characterized, we propose that the presence of its
based amphiphiles have favorable physicochemical properties of mesomeric structures can be used as a potential phosphate
lipids, such as formation of stable liposomes or giant unilamellar bioisostere.8-11 The squaramide moiety has extensive
vesicles in aqueous solution, high phase-transition temperature, applications including bioisosteres for various acidic functional
10
low vesicle leakage and phospholipase resistance properties.
groups.9, A varieties of squaramide-based compounds are
used in chemistry and medicinal chemistry related applications.
Recently, squaramide containing oligonucleotide analogues
were synthesized and incorporated into the DNA duplexes.
Squaramide-based other biomolecules like 2′-deoxynucleotides
and ribonucleotides, pyrophosphate mimic were also
reported.9-11
Naturally occurring phospholipids contain headgroup with
phosphate moiety at the bilayer interface and charged or
neutral moiety extended into the aqueous phase. The liposome
formation ability of these phospholipids has been widely used
for drug delivery, artificial cell construction and others.1-3
Phospholipids are also used as therapeutic agents, like
pulmonary surfactant.3, 4 These significant biological functions
and pharmaceutical applications of the natural phospholipids
have incited researchers to develop several unnatural lipids to
improve properties of lipids and diversify its applications.1-3, 5-7
Surprisingly, all the efforts have been concentrated on
modifying the headgroups, hydrophobic tails and the linker
region between the headgroup and the tails of the lipid, without
altering the phosphate moiety. Recently reported inverse-
phosphatidylcholine with inverted charge orientation in the
choline headgroup leads to a number of interesting and
complimentary properties in lipids.2 In this report, we
investigate how the presence of a phosphate bioisostere can
alter the physicochemical properties of the lipid bilayer in
comparison with the phospholipids. We hypothesize that the
uses of phosphate bioisostere containing lipids can be
advantageous because of its probable interference with the
phosphate-based cellular processes and crucial for obtaining
better or tunable properties.
In this regard, synthesis of membrane forming lipids that
contain squaramide moiety in the place of a phosphate group
of the phospholipids might help to understand their physical
properties under the liposomal environment or provide new
member for liposome-based therapies are of great importance.
It is possible that the presence of squaramide group can perturb
the phospholipase activities and may prolong stability of the
liposomes. Therefore, SA lipids could have several applications
in liposomal drug delivery, formation of model membrane and
others.
We synthesized a family of squaramide-based (SA) lipids
with choline, serine and glycerol-like headgroups that extend
into the aqueous phase and a squaramide moiety adjacent to
the lipid bilayer (Scheme 1). For all these lipids saturated and
a. Department of Chemistry, Indian Institute of Technology Guwahati, Assam
781039, India.; E-mail: dmanna@iitg.ernet.in
‡ Contributed equally to this body of work.
Electronic Supplementary Information (ESI) available: Experimental procedures,
synthetic methods and supplementary figures are provided. See
DOI: 10.1039/x0xx00000x
Scheme 1 Synthetic rout to a novel series of squaramide-based lipids.
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showed a smaller melting range for the membrane like model
unsaturated alkyl chains were connected via ester linkages lipid DPPC, indicating better cooperativity during phase
SA1-8). To understand the role of ester linkage on the SA lipids, transition (Fig. 1A). The Tm values of the unsaturated lipids (SA5-
we also synthesized ether based lipid, SA9 with saturated alkyl ) could not be measured because of the lowest temperature
(
8
chain and choline like headgroup. Condensation of ester or setup for our fluorimeter was 5 °C, but we presume that the Tm
ether-linked long-chain amine with N-substituted derivatives of value of the unsaturated SA lipids will be lower than the
3-amino-4-ethoxycyclobut-3-ene-1,2-dione or squaric acid saturated ones. The reported Tm values of the unsaturated lipids
yielded the target SA lipids in a single step with good yields.
like DOPC, DOPS and DOPG are < −10 °C.16 For ether lipid, SA9
Transition from gel to liquid phase is considered as an the Tm value was 55 °C, which is comparable with that of SA1
essential physical property of the lipids to investigate the lipid (Fig. S1, ESI†). Additional microviscosity studies under the
thermal stability and drug release profile of the liposomes.2, 3 liposomal environment revealed that the microviscosity
The phase transition temperature (Tm) values of the lipids parameter of DPH decreases with the increase in temperature
primarily depend on the inherent properties of headgroup, and correlates well with its fluorescence anisotropy values
saturation/unsaturation and length of the hydrophobic alkyl (Table S1, ESI†).17 This study suggests that the DPH molecules
chain.2, 12 Temperature dependent steady-state anisotropy of experienced lesser internal resistance (below Tm values) under
the membrane embedded- 1,6-diphenyl-1,3,5-hexatriene (DPH) the liposomal environment of SA1/SA9 lipids than the DPPC
fluorescence probe was employed to determine the Tm values lipid (Fig. S2, ESI†). These differences in microviscosity
of the of the membrane bilayer generated from the SA lipids.3 parameters may have functional significance in the regulation
Large unilamellar vesicle (LUV) was used for this study. The of molecules across the bilayer of SA lipids.
observed Tm values of SA1
66 and 39 °C, respectively; which are significantly different than transmission electron microscopy (TEM).2,
the corresponding phospholipids (Fig. 1B and Fig. S1, ESI†). The revealed predominant formation of small and medium sized
observed elevation of Tm values (by 10-20 °C) for SA1-3 lipids in liposomes by the saturated (SA2-4) and unsaturated (SA6-8
comparison with the corresponding phospholipids (of same lipids. The SA1 SA5 and SA9 lipids predominantly forms
,
SA2
,
SA3 and SA4 lipids were 51, 68,
Liposome formations by the SA lipids were verified by
14
The images
)
,
chain length) suggest that the presence of squaramide liposomes, but also form small amount of other lipid aggregates
containing headgroups have a substantial effect on the phase (Fig. 2B-F and Fig. S3, ESI†) which could be due to their less
transition and may have more thermal stability and stronger hydration capabilities in the aqueous environment or their
drug retention ability than natural phospholipids.13-15 The interaction with the cations present in the buffer. The
presence of squaramide moiety can increase the lipophilicity of interaction of cation with the squaramide moiety could enhance
the SA lipids, leading to a more ordered packing within the lipid the aromaticity and stabilization (Fig. S4, ESI†) of the
bilayer. The squaramide
0.4
0.35
0.3
80
70
60
50
40
30
20
10
0
(A)
(B)
0.25
0.2
SA1
DPPC
0.15
0.1
0.05
0
DPPC
SA1
SA2
SA3
SA4
SA9
0.28
0.3
1/T x 102
0.32
0K-1
0.34
0.36
(
)
Fig 1. The Tm of SA lipids by steady-state anisotropic measurements. (A)
Anisotropic curve of SA1 and DPPC lipids within the range of 5 – 90 °C. (B) Tm
values of the saturated SA lipids. Error bars denote standard deviation.
Fig. 2 (A) General structure of the SA lipids. (B-F) TEM image of the
liposomes generated from 100% SA lipids, scale bar = 200 nm.
moiety can act as both hydrogen bond donor and acceptor.10, 11
Therefore, interaction among the squaramide moieties of the
lipids can also enhance the Tm values of the SA1-3 lipids.10 These
higher Tm values suggest that the headgroups of the SA1-3 lipids
may have less hydration capabilities than the corresponding
phospholipids. Lower Tm value of SA4 lipids than the other SA
lipids suggests that either it has more hydration capability or its
squaryl monoamide moiety (remains in monoanionic form at pH
7.2) interacts differentially at the bilayer surface. Wider melting
ranges for the SA2-4 lipids also suggest that these lipids are less
cooperative during phase transition.3 On the contrary, SA1 lipid
SA1,
SA5 and SA9 lipids.10 Stabilization of counter anion by the
secondary amine and trimethylethanaminium group of the
squaramide moiety could also assist the interaction of cation
with these lipids. Addition of these SA lipids to a mixture of
model lipids like DPPC, DPPS and cholesterol (in the molar ratio
of 2:4:2:2) also showed excellent liposome formation
capabilities (Fig. S5, ESI†). This indicates the capability of SA
lipids in forming liposome with other phospholipids, which
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could be beneficial for the preparation of size tunable liposomes moiety.10 This experiment also shows no significant effect of the
and other specific uses. differently charged-headgroups (choline, serine and glycerol-
The potential of these SA lipids to form giant unilamellar like moieties) on the overall surface potential of the lipids. The
vesicles (GUVs) was investigated by electroformation method.18 small differences in the surface potential values among the
GUVs with ≥ 10-15 nm diameters are considered as a model lipids could be because of the effect of headgroups on the
liposomes for mimicking the cellular membrane because of mesomeric structures of squaramide.10 Simultaneous dynamic
their comparable size of the mammalian cells and the ability to light scattering (DLS) measurements of the liposomes at
act as physical barriers for the encapsulated ingredients.3 different pH showed that the changes in the diameter of
Fluorescence microscopic images showed that 100 % SA1 and liposomes of SA lipids are negligible (Fig. S7, ESI†).
SA9 lipids are capable of forming excellent GUVs (Fig. 3 and Fig.
Divalent cations often interact with the lipid headgroups to
S6, ESI†). Encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid modify surface potential or induce stability, fusion and
(HPTS) and membrane embedded Texas Red 1,2- aggregation of the liposomes.2, 14 In this regard, we measured
dihexadecanoyl-sn-glycero-3-phosphoethanolamine
(Texas- the effect of Ca2+ on the surface potential of the liposomes of SA
Red DHPE) were used as a fluorescent marker for an aqueous lipids. The zeta potential values of the SA1-4 lipids (at pH 7.2)
environment and to visualize the membrane, respectively. The increase with increasing concentration of Ca2+ ion, but remain
GUV formation properties of the SA lipids clearly demonstrate negative (up to 5 mM Ca2+ concentration). This slight increase in
their capability of forming biomimetic membranes, which can surface potential of the SA lipids indicates that Ca2+ ion weakly
be used as model system for living cells for mimicking cellular interact with the liposomal surface (Fig. 4B). DLS measurements
processes.
of the liposomes in the presence of Ca2+ ion showed negligible
changes in the diameter of liposomes of SA lipids (Fig. S8, ESI†).
This weak and/or nonspecific interaction of the SA lipids with
the Ca2+ ion could be advantageous for its drug delivery
applications.2
The charged headgroups of the lipids have the inclination to
attract charged molecules or ions to the surface or core of the
liposomes, which results in an increase in bilayer permeability
of the charged molecules or ions.14 In this regard, the
permeability
of
water-soluble
anionic
reporter
carboxyfluorescein (CF) and cationic reporter rhodamine 6G
(R6G) through the liposomes was measured using vesicle
Fig. 3 Fluorescence microscopic images of the GUVs generated from 100 %
SA1 lipid. (A) Bright field. (B) Green channel illustrating HPTS encapsulation.
(C) Red channel illustrating the staining of the lipid membrane with Texas-Red
DHPE. (D) Merge of green and red channels.
Due to the presence of squaramide containing zwitterionic,
cationic and neutral headgroups, we investigated the overall
surface potential of the liposomes (LUV) of the SA lipids.2, 14 The
PH-dependent zeta-potential measurements of the SA1-4 and
SA9 lipids revealed that all these lipids have negative surface
potential at higher pH (Fig. 4A). The negative surface potential
of these lipids decreases with decrease in pH. We presume that
the surface potential of the unsaturated lipids (SA5-8) would be
of very similar pattern because of the presence of same
headgroups. At lower pH, SA lipids are slightly negative than
DPPC, which suggest that pKa of SA lipids (< 3.5) may be lower
than DPPC but higher than DPPS, DPPG and DPPA lipids. The
surface potential measurements also revealed that the charge
properties of SA lipids are significantly different from the tested
Fig. 4 (A) Zeta potential of the liposomes at different solution pH. (B) Shift in
zeta potential in the presence of Ca2+ ion. (C) CF and (D) R6G release profiles
from the liposomes of 100 % SA lipids. Error bars denote standard deviation.
°
leakage assay at 37 C. The extents of CF and R6G release
14
anionic lipids but have subtle difference with DPPC.2, The
through the liposomes of pure SA1-3, SA9 lipids are low (20-40
negative surface potential of these lipids in higher pH solution
could be due to high aromatic stabilization of the squaramide
% release after 60 hours), indicating a weak interaction of the
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CF and R6G molecules with the liposomal surfaces of the phospholipase resistance properties of the SA lipids would
corresponding SA lipids (Fig. 4C, D). These SA lipids also showed greatly benefit the research and development of a novel lipid.
more than two-fold lower release rate in comparison with the
In conclusion, we developed a novel class of ester and ether lipids
corresponding phospholipids under the similar experimental with squaramide moiety as phosphate bioisostere. These SA lipids
conditions (Fig. S9, ESI†). Therefore, our microviscosity studies also contain choline, serine and glycerol-like headgroups. High
and vesicle leakage analyses suggest that headgroup of the SA phase transition temperature, ability to form liposomes and giant
lipids may plays an important role on the membrane unilamellar vesicles in aqueous solution, slow leakage profile for
permeability of the molecules. A slightly higher amount of R6G cationic and anionic molecules and phospholipase resistance are the
release through the liposomes of pure SA4 lipid could be due to salient features of these squaramide-based lipids. These lipids show
its interaction with the SA4 headgroup (Fig. 4D). Unsaturated negative surface potential at higher pH and do not significantly
SA lipids also showed a similar vesicle leakage profile in the interact with Ca2+ ion. Therefore, these SA lipids provide a powerful
presence of both CF and R6G molecules (data not shown). The tool for the applications of developing liposomal drug delivery
liposomes of mixed lipids of SA1-4 or SA9 with systems, artificial cells and others. These SA lipids also provide
DPPC/DPPS/cholesterol (molar ratio of 2:4:2:2) also showed additional opportunities to study the influence of both hydrogen
slightly higher release rate for R6G molecules (Fig. S9, ESI†). donor and acceptor properties of the squaramide moiety on the
However, cholesterol has a massive effect on the membrane membrane dependent intrinsic biological activities.
fluidity and therefore lower any transmembrane transport.19
We acknowledge the financial support from DST (SB/FT/CS-
So, the higher release rate of R6G through the liposomes of the 131/2012), New Delhi and Central Instrument Facility, IIT
mixed lipids could be due to the presence of DPPC and DPPS Guwahati for the instrumental facility.
lipids. This result suggests that other lipids can be used to tune
the content release profile for the positively charged molecules
Notes and references
1. Z. Huang and F. C. Szoka, J. Am. Chem. Soc., 2008, 130
15702.
2. E. K. Perttu, A. G. Kohli and F. C. Szoka, J. Am. Chem. Soc.,
2012, 134, 4485.
3. C. Y. Zhou, H. X. Wu and N. K. Devaraj, Chem. Sci., 2015,
4365.
4. M. Griese, European Respir. J., 1999, 13, 1455.
5. Y. J. Rui, S. Wang, P. S. Low and D. H. Thompson, J. Am.
Chem. Soc., 1998, 120, 11213.
6. B. Jing, N. Tokutake, D. H. McCullough and S. L. Regen, J. Am.
Chem. Soc., 2004, 126, 15344.
7. M. M. Ma, Y. Gong and D. Bong, J. Am. Chem. Soc., 2009,
131, 16919.
8. K. Seio, T. Miyashita, K. Sato and M. Sekine, Eur. J. Org.
Chem., 2005, 24, 5163.
9. J. Aleman, A. Parra, H. Jiang and K. A. Jorgensen, Chem. Eur.
J., 2011, 17, 6890.
10. R. I. Storer, C. Aciro and L. H. Jones, Chem. Soc. Rev., 2011,
40, 2330.
11. T. S. Elliott, A. Slowey, Y. L. Ye and S. J. Conway, Med. chem.
comm., 2012, 3, 735.
12. F. Sixl and A. Watts, Proc. Natl. Acad. Sci. USA, 1983, 80
1613.
13. V. J. Venditto, A. Dolor, A. Kohli, S. Salentinig, B. J. Boyd and
F. C. Szoka, Chem. Comm., 2014, 50, 9109.
14. N. Mamidi, S. Gorai, B. Ravi and D. Manna, Rsc Adv., 2014,
21971.
of the SA lipids. The adjustable release rate could be useful in
developing new approaches for liposome-based drug delivery
methods. However, additional biophysical studies are required
to understand if this is a general property of the SA lipids.
Additional R6G release profiles of 100 % SA1-4 and SA9 lipids
containing liposomes were performed in the presence of Ca2+
ion (Fig. S10, ESI†). The results revealed that Ca2+ ion
concentrations up to 5 mM have no considerable effect on the
R6G release profile of these lipids; which is another important
characteristic for the stability of these liposomes under
physiological conditions. These vesicle-leakage data suggest
that SA lipid containing liposomes may be useful for
encapsulating and delivering water soluble drug molecules.
Another potential advantage of these lipids is their
enhanced stability towards phospholipases, which generally
hydrolyze phospholipids under the physiological conditions.
The phospholipases are commonly found in mammalian tissues
and plays an important role in the inflammatory responses.3, 20
Enzymatic assays clearly showed that phospholipase-A2 (PLA2)
and phospholipase C (PLC) enzymes had no hydrolytic activity
against SA1 and SA9 lipids (Fig. S11-13, ESI†). However, both
PLA2 and PLC enzymes successfully hydrolyzed DPPC lipid under
the similar experimental conditions. The PLA2 family of
enzymes generally hydrolyzes the sn-2 acyl-bond of the diacyl-
glycerophospholipids.3, 20 SA1 lipid contains sn-2 acyl-bond but
SA9 lack ester bonds. Therefore, the presence of squaramide
moiety devises these lipids proficient enough to resist from PLC
and PLA2 dependent hydrolysis, which indicates its resistibility
from phosphlipase activity. We presume that liposome/GUV
composed of SA1-9 and natural phospholipids can be used in
combination in the presence of PLA2 enzyme to tune the
shrinkage of the size of liposome/GUV, which has wider
applications in imaging, therapeutics and others. We envision
faster installation of squaramide moiety into the parent lipid
scaffold, for easy access to introduce several functionally with
high yields. Therefore, we foresee that the rapid synthesis and
,
6,
,
4,
15. E. K. Perttu and F. C. Szoka, Chem. Comm., 2011, 47, 12613.
16. J. R. Silvius, Lipid-Protein Interactions, 1982, John Wiley &
Sons, Inc., New York.
17. J. Swain, M. Kamalraj, H. S. P. Rao and A. K. Mishra, Rsc Adv.,
2014, , 55377.
4
18. C. Herold, G. Chwastek, P. Schwille and E. P. Petrov,
Langmuir, 2012, 28, 5518.
19. E. M. L. Bastiaanse, K. M. Hold and A. VanderLaarse,
Cardiovas. Res., 1997, 33, 272.
20. E. A. Dennis, J. Biol. Chem., 1994, 269, 13057.
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