Aggregation Behavior of Non-ionic Twinned Amphiphiles and Their Application as Biomedical Nanocarriers

Article abstract of DOI:10.1002/asia.201700450

A new class of twinned amphiphiles was developed by conjugating a pair of hydrophilic head groups from mPEG chains (M_n: 350 or 1000) and a pair of hydrophobic segments from linear alkyl chains (C₁₁ or C₁₈) through a novel spacer synthesized from glycerol and p-hydroxybenzoic acid. The aggregation phenomena of the amphiphiles were proven by DLS and fluorescence experiments, whereas size and morphology of the aggregates were evaluated by cryo-TEM. The measurements proved the formation of globular, thread-like or rod-like micelles as well as planar double-layer assemblies, depending on the amphiphile's molecular structure. The applicability of these non-ionic amphiphilic systems as nanocarriers for hydrophobic guest molecules was demonstrated by encapsulating a hydrophobic dye, Nile Red, and a hydrophobic drug, Nimodipine. The transport capacity results for both Nimodipine and Nile Red prove them as a promising candidate for drug delivery.

Full text of DOI:10.1002/asia.201700450

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Aggregation behaviour of non-ionic twinned amphiphiles and their
application as biomedical nanocarriers
Authors: Abhishek K. Singh, Bala N. S. Thota, Boris Schade, Katharina
Achazi, Abdullah Khan, Christoph Böttcher, Sunil K. Sharma,
and Rainer Haag
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Chemistry - An Asian Journal
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Aggregation behaviour of non-ionic twinned amphiphiles and
their application as biomedical nanocarriers
[
a]
[b]
[c]
[b]
[a]
Abhishek K. Singh , Bala N. S. Thota , Boris Schade , Katharina Achazi , Abdullah Khan ,
[
c]
[a]
[b]
Christoph Böttcher* , Sunil K. Sharma* , Rainer Haag*
natural analogs.6-10 Recently, Zhu et al. developed a drug self-
delivery system for cancer therapy by synthesizing amphiphilic
drug-drug conjugate.11
Abstract A new class of twinned amphiphiles was developed by
conjugating a pair of hydrophilic head groups from mPEG chains
(Mn: 350 or 1000) and a pair of hydrophobic segments from linear
alkyl chains (C11 or C18) through a novel spacer synthesized from
glycerol and p-hydroxybenzoic acid. The aggregation phenomena of
the amphiphiles were proven by DLS and fluorescence experiments,
whereas size and morphology of the aggregates were evaluated by
cryo-TEM, the measurements proved the formation of globular,
thread-like or rod-like micelles as well as planar double-layer
assemblies, depending on the amphiphile’s molecular structure. The
applicability of these non-ionic amphiphilic systems as nanocarriers
for hydrophobic guest molecules was demonstrated by
encapsulating a hydrophobic dye, Nile red, and a hydrophobic drug,
Nimodipine. The transport capacity results for both Nimodipine and
Nile Red prove them as a promising candidate for drug delivery.
Among SMAs, Gemini amphiphiles have attained considerable
attention, due to their superior physicochemical properties such
as low critical aggregation concentration (CAC) and the
formation of stable aggregates.12-14 The influence of different
molecular parameters on the aggregation behaviour of Gemini
amphiphiles is well reported in the literature15,16 and the
knowledge of such dependencies has been utilized for the
design of novel carriers in biomedical applications, particularly in
gene delivery as most of the Gemini amphiphiles reported so far
are cationic in nature.17-19 There are, however, only few reports
available describing their applications as nanocarrier for the
delivery of hydrophobic drug molecules. A major reason for this
Introduction
Self-assembly of amphiphilic molecules is a prevalent principle
of nature to construct functional entities covering diverse
phenomena like protein folding, compartmentalization, and
information transcription. Although amphiphilic molecules have
been studied for decades to understand their physicochemical
properties and aggregation behaviour, only recently controlled
architectures are studied for potential applications for drug /
gene delivery and in optoelectronics among others.1,2,3 So far, a
large number of amphiphilic systems have been developed and
investigated for their self-assembly into a wide variety of
nanostructures.4,5 However, these can be broadly divided into
two major classes each comprising its own advantages. While
polymeric amphiphiles (PA) result in more stable nanostructures,
small molecule amphiphiles (SMAs) are more simple in design
and their self-assemblies displays more similarity with the
may be that the cationic Gemini amphiphiles are known to
acquire cytotoxicity20 while particularly non-ionic amphiphiles are
advantageous for drug delivery applications.21,22 Non-ionic
Gemini amphiphiles based on carbohydrates have been well
studied in literature.23-26 However, our interest is to explore the
use of other competing moieties to come up with an amphiphilic
system with an improved capacity for drug delivery. In this
respect, polyethyleneglycol (PEG) is considered as a potential
choice due to its specific properties, such as water solubility,
non-toxicity, decreased interaction with blood components, good
chemical stability, and biocompatibility. Implementing non-ionic
PEG as the hydrophilic building block of Gemini amphiphiles
provides a new type of amphiphiles featuring the beneficial
characteristics of pegylation like biocompatibility, reduced
immunogenicity and antigenicity, as well as prolonged circulation
by reduced renal clearance.27,28 The availability of PEG in a
broad range of molecular weights is additionally advantageous
to fine-tune the properties of the such amphiphiles. Although the
[
[
a]
b]
Abhishek K. Singh, Abdullah Khan, Prof. Sunil K. Sharma
Department of Chemistry, University of Delhi, Delhi, India
E-mail: sksharma@chemistry.du.ac.in
B.N.S. Thota, Dr. Katharina Achazi, Prof. Rainer Haag
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße
physicochemical behaviour of PEG-based Gemini amphiphiles
has been studied before29-31, only few such molecules have
3, 14195 Berlin, Germany
E-mail: haag@zedat.fu-berlin.de
[
c]
Dr. Boris Schade, Dr. Christoph Böttcher
Forschungszentrum für Elektronenmikroskopie, Institut für Chemie und
Biochemie, Freie Universität Berlin, Fabeckstraße 36a, 14195 Berlin,
Germany
been applied as nanocarriers for hydrophobic molecules.32,33
To expand the scope of such systems for drug delivery
applications, we have synthesized a series of new non-ionic
amphiphilic compounds (1-4) (Figure 1), which are constituted
from two hydrophilic (mPEG) and two hydrophobic (alkyl chain)
E-mail: christoph.boettcher@fzem.fu-berlin.de
Supporting information for this article is given via a link at the end of the
document. ((Please delete this text if not appropriate))
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moieties connected via a triazolyl p-hydroxybenzoate spacer
arrangement of the paired hydrophobic and hydrophilic
segments around the shared spacer.35
(
2 2
Scheme 1). This is the first report of using such an A B spacer
moiety with aromatic and non-aromatic units that may enhance
the solubilization and encapsulation potential of the synthesized
amphiphilic units for hydrophobic cargo delivery.21,34 We termed
the compounds twinned because they do not exactly fit to the
definition of Gemini amphiphiles, having a slightly different
The new amphiphiles were characterized with respect to their
assembly behaviour by using fluorescence, dynamic light
scattering (DLS), and cryogenic electron microscopy (cryo-TEM).
Furthermore, their application as nanocarriers for hydrophobic
guests is demonstrated by using two established standards, the
hydrophobic dye Nile red and the hydrophobic drug Nimodipine.
Scheme 1. Synthesis of mPEG based non-ionic twinned amphiphiles: a(i) EDC.HCl, DMAP, DCM, 15 h, 30 oC; b(ii) KMnO
, NaOH, water, 24 h 80 oC; c(iii)
4
Novozym 435, vinyl acetate, THF, 2 h, 30 C; (iv) ethyl 4-hydroxybenzoate, DIAD, TPP, THF, 15 h, 30 oC; (v) 1N NaOH in methanol, water, 4 h, 50 C; (vi)
o
o
o
o
propargyl bromide, K
2 3
o
CO , DMF, 6 h, 60 C; (vii) compound 5/6, sodium ascorbate, copper sulphate, THF:water (3:1), 15 h, 50 C; (viii) compound 7/8, EDC.HCl,
DMAP, DCM, 15 h, 30 C.
Results and Discussion
Synthesis
(Mn: 350 or 1000) chain lengths (Scheme 1). All of these
amphiphiles were synthesized starting from glycerol which was
first converted to 2-hydroxypropane-1,3-diyl diacetate using vinyl
The aggregation behaviour of amphiphilic system depends on
several factors such as the length of the different structural
constituents (hydrophilic, hydrophobic moieties and spacer). To
investigate the influence of the size of the hydrophobic alkyl
moiety and the chain length of the hydrophilic mPEG group on
the aggregation behaviour, we have synthesized four twinned
amphiphiles (1-4) by varying the alkyl (C18 or C11) and mPEG
acetate as the acylating reagent, by following the established
bio-catalytic procedure.36 The secondary hydroxyl group of 2-
hydroxypropane-1,3-diyl diacetate was then coupled with 4-
hydroxybenzoate via Mitsunobu reaction.37 The resulting
1
compound 9 was characterized from its spectral data. In its H
2 2
NMR spectrum, the methine (-OCH(CH O-) ) protons get
deshielded and appear at δ 4.79-4.75 as compared to the
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precursor glycerol 1,3-diacetate (δ 4.1). Hydrolysis of the
product with methanolic NaOH solution yielded compound 10.
in Milli Q water was prepared and the pyrene stock solution was
used to prepare different samples of the amphiphiles with
The characteristic signals for the acetyl moiety i.e. the singlet at
δ 2.06 in H NMR and the peaks at δ 20.86, 170.79 in the 13
1
C
NMR spectra in compound 9 disappeared completely upon
hydrolysis. Compound 10 was further subjected to selective
propargylation at the carboxylic site to obtain the desired p-
hydroxybenzoate spacer 11. This compound was characterized
on the basis of its 1H-NMR spectrum, the methylene (-
2
2
CH C≡CH) and methine (-CH C≡CH) protons of the propargyl
moiety appear at δ 4.86 and 2.50, respectively. Diacyl 2-azido
glycerol (5/6) was then “Click“ coupled at the propargyl end of
the spacer forming compounds 12 and 13. Finally, mPEG acid
(
7/8) was linked to the diol (12 /13) via esterification to obtain the
targeted twinned amphiphiles (1-4) (Scheme 1).
Characterization of these amphiphiles using 1H- and 13C-NMR,
IR, and gel permeation chromatography (GPC) (Figure 1)
unambiguously validated their intended structure. The
characteristic triazolyl ring protons appear in the range δ 7.77-
Figure 1. Calculation of HLB value using Griffin equation.
required concentrations. All the solutions were filtered using 0.22
μm polytetrafluoroethylene (PTFE) filter to remove the non-
encapsulated dye. The fluorescence spectra were recorded at
7
.78 in the 1H NMR spectra, while the methylene protons
o
neighbouring the triazole ring appear at δ 5.0-5.5 ppm. The
molecular weight and PDI for all of the resultant amphiphiles (1-
20 C for the filtered clear solutions and from the fluorescence
1 1 3 3 3 1
intensity data, I (λ : 372 nm) and I (λ : 385 nm), the ratio of I /I
4
) obtained from GPC are listed in the Figure 1.
was calculated and plotted with the log [amphiphile
concentration] to obtain the CAC values (Figure 2). Since the
amphiphile 1 has limited solubility in aqueous medium, the CAC
values were recorded for amphiphiles 2, 3 and 4 only.
Aggregation behaviour
Four different non-ionic twinned amphiphiles (1-4) synthesized
by varying the ratio of hydrophilic and hydrophobic moieties
were observed to exhibit variation in solubility and aggregation
behaviour in aqueous medium. Amphiphiles 3 and 4 having
larger mPEG (Mn: 1000 g/mol) turned out to be more soluble in
water as compared to 1 and 2 with smaller mPEG units (Mn: 350
g/mol), this behaviour can be quantified by the Griffin method of
evaluating the hydrophilic-lipophilic balance (HLB) for non-ionic
surfactants (see Figure 1).38 The aggregation behaviour of the
amphiphiles (1-4) was studied in aqueous solution by
fluorescence spectroscopy, dynamic light scattering (DLS), and
cryogenic electron microscopy (cryo-TEM). The CAC of
amphiphiles was measured by fluorescence spectroscopy using
pyrene as a probe. In general, the emission spectrum of pyrene
displays a well-defined vibronic structure. The intensity of the
first and third vibronic peaks is considerably sensitive to the
polarity of the surrounding medium and their ratio is a good
measure for the microenvironment of the fluorophore.39 In our
typical experimental setup, a stock solution of pyrene (0.5 mM)
0.75
0.70
0.65
2
3
4
0.60
0.55
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Concentration (% wt.)
Figure 2: Determination of the critical aggregation concentration of
amphiphiles 2-4 using fluorescence spectroscopy and pyrene as a probe.
Amphiphile 3 constituted from larger hydrophobic (C18 alkyl
chain) and hydrophilic (mPEG Mn: 1000) groups was found to
have lowest CAC value of 0.7 x 10-5 M. However, reducing the
size of hydrophobic alkyl chain (C ) increases the CAC by two
1
1
fold in amphiphile 4 with a value of 1.4 X 10-5 M. On the other
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hand reducing the size of both, hydrophobic (C11
)
and
arrowheads). As this latter morphology reflects the different
orientation at a 360° turn it clearly identifies the ribbon-like
nature of the assemblies. Broader ribbons indicate a more
pronounced tendency towards lateral growth instead of stacking
(black arrowhead). Sometimes they widen in a stepwise manner,
suggesting their construction from side by side arranged narrow
ribbons. (see also SI for TEM micrographs of negatively stained
samples).
hydrophilic (mPEG Mn: 350) groups enhances the CAC value
significantly to 4.8 x 10-5 M.
The size of the aggregates was determined by dynamic light
scattering (DLS) measurements. For amphiphiles 1-3, DLS
displayed unimodal size distribution of the formed aggregates
with diameters of around 85, 53, and 98 nm, respectively, while
the least hydrophobic compound 4 exhibited a bimodal intensity
profile with peaks at 11 and 125 nm. Corresponding plots of the
volume distribution displaying values of 53, 35, 40 and 9 nm for
amphiphiles 1, 2, 3 and 4, respectively (Figure 3), clearly
indicate that smaller aggregates represent the majority of
entities present within the samples.
25
20
15
10
5
0
1
2
3
4
Figure 4. Cryo-TEM micrograph of amphiphile 1 at a concentration of 0.125
wt%. Planar ribbons of different width and length dominate the preparation
(
(
black arrowhead) and occasionally provide their contrast bilayered side views
black-rimmed arrowheads).Twisted entities (white arrowheads) identify the
complete morphology of the assemblies. Bar is 200 nm.
Both amphiphiles
2 and 3 form elongated assemblies in
1
10
100
Size (d nm)
1000
10000
aqueous solutions (Figure 5). Amphiphile 2 self-assembles into
flexible threads with diameters ranging from 4.7 to 5.5 nm which
appear as individual aggregates even at a concentration as high
as 0.5 wt%. Dark spots along the course of the threads
represent top views of sections that are situated perpendicular to
the embedding ice film.
Figure 3: DLS profile for the amphiphiles 1-4 in water.
Cryo-TEM
The morphology of the twinned amphiphiles aggregates was
studied in aqueous solutions using cryogenic transmission
electron microscopy (cryo-TEM). All samples were prepared
according to our standard preparation protocol (see SI). Since
amphiphile 1 has limited solubility in pure water, self-assembly
of this compound was investigated in the presence of small
amount of THF (2.5% v/v). At an amphiphile concentration of
Samples of amphiphile 3 showed individual assemblies only at
concentrations of 0.125 wt%. Moreover, these assemblies
appear relatively stiff and have diameters of around 8 - 9 nm.
Also, a less contrast central channel can be observed at several
well resolved entities. As a result of their stiffness most of the
aggregates were found densely adjoined. Cross-sectional
circular views like those described for the aggregates of 2 (cp.
above) could not be observed here because the rods are too
long and too rigid to become orientated perpendicular to the thin
0
.25 wt%, individual aggregates could hardly be distinguished
due to the dense packing. At reduced concentration of 0.125
wt% individual flat and also twisted ribbons of several 100 nm in
length could be identified (Figure 4). In some cases flat ribbons
present their side views with a typical bilayer density profile of
about 8 - 9 nm thickness (black-rimmed arrowhead). Twisted
ribbons, which occasionally occur, provide sequences of high-
contrast side views and low contrast top views (white
(
100 to 300 nm) ice films. Nevertheless, from the uniform
thickness of the aggregates a circular profile can be assumed.
This assumption became quite more evident when the sample
was prepared in an aqueous mixture with 10% (v/v) of methanol
(
see SI).
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globular micelles but none of the larger aggregates were
observed (Figure 6C), providing strong evidence that the
hexagonal arrays originate rather from a dense arrangement
due to high local concentration of assemblies than from
assembly interactions.
Figure 5. Cryo-TEM micrographs of amphiphiles 2 and 3 which both form
fibrous aggregates in water. According to the hydrophobic length of the
constituting compounds they strongly differ in diameter and flexibility. Twinned
amphiphile 2 forms flexible micellar threads with 5 nm in diameter (A) of which
frequently cross sectional top views can be recognized as dark spots along the
threads course (white arrowheads), while its homologue 3 assembles into
thicker (~8.5 nm) and more rigid rods (B). Bars represent 100 nm.
Herein, the number of aggregates was reduced due to the
enhanced solubility and individual rods with diameters of about 9
nm were visible. However, groups of parallel rods were
frequently found giving the opportunity to compare the rod
morphology from both solvents in detail. Obviously, minor
changes of the solvents’ polarity do not significantly affect the
overall architecture of the emerging aggregates and thus
justifies the use of solvent mixtures slightly differing in polarity,
e.g. for amphiphile 1.
Figure 6. Cryo-TEM images of the 0.5 wt% aqueous solution of 4 exhibit
spherical micelles of 14 to 18 nm (central region) next to featureless large
aggregates of different diameters (black-rimmed arrowheads) (A) Arrays of
densely packed micelles which sometimes present an ideally hexagonal
arrangement (B) display sharp peaks in the Fourier transform (inset), which
correspond to 8.2 ± 0.3 nm distances in real space. Dissolution of the sample
to 0.25 wt% gives individual micelles (C).
Micrographs of vitrified samples of the best water-soluble
amphiphile 4 (0.5% wt in pure water) displayed two different
kinds of assembly structures (Figure 6A), i.e. (i) spherical
entities with a relatively narrow sizes distribution of 14 to 18 nm
Bars represent 200 nm (A) or 100 nm (B and C).
(
central region in Figure 6A) tending to form highly ordered
The microscopic data proof a marked relationship between
molecular and supramolecular structure for the reported series
of compounds. The increase of curvature starting with two-
dimensional ribbons (1) via one-dimensional fibers and rods (2
and 3, respectively) into globular micelles (4) is in line with the
arrays and (ii) markedly larger smooth unstructured structures of
higher contrast with different diameters up to the micrometer
scale (black-rimmed arrows). FFT of the densely packed arrays
allowed for
a
precise determination of the aggregates
dimensions. The sharp reflexes in the Fourier transform (inset in
Figure 6B) assign ideally hexagonal arranged entities of very
uniform repeat distances of 8.2 ± 0.3 nm. Given the size and the
noticeable monodispersity, these aggregates have to be
considered as globular micelles.
concept of
a hydrophilic-hydrophobic balance defining the
assembly curvature according to Israelachvili et al.40,41 i.e.
amphiphile 1 with long hydrophobic tails and short hydrophilic
head groups forms rather planar aggregates with low curvature,
whereas amphiphiles 2 and 3 with their “balanced” ratio of
hydrophobic and hydrophilic constituents (short tails and heads
or long tails and heads, respectively) are forming micellar fibres
of moderate curvature (Figure 7, top and centre). Amphiphile 4
with short tails and large head groups assembles into small
spherical aggregates of highest curvature (Figure 7, bottom).
The micelle arrays feature sharp boundaries against the
surrounding vitrified solvent giving the impression of attractive
interactions between the micelles (white arrow heads in Figure
6
A). To further investigate the extent of assembly interactions,
cryo-TEM measurements of a diluted sample of 4 at 0.25% (wt)
in water were performed. Hereby, individually distributed
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Thus, it can be stated that the classical concept of the
interdependence of assembled architecture and packing
parameter also holds for this group of compounds with their
flexible twinned head groups and tails and their bi-aromatic
spacer. Moreover, here the longer hydrophobic tails of 1 and 3
account not only for thicker and broader ribbons and rods but
also for obviously more rigid assemblies, if compared to the
flexible threads from amphiphile 2.
Modelling of the twinned amphiphiles provided evidence for the
molecular construction of the observed aggregates. If a bilayer
(1) or bimolecular (3) arrangement of the molecules is assumed,
both the dimensions (vide supra) coincide fairly well with the
length of the hydrophobic parts of the constituting twinned
amphiphiles (i.e. the distance between the terminal hydrogen
and the carboxylic oxygen of the head group binding ester
group) which is 3.8 and 3.0 nm, respectively (Figure 1).
Furthermore, the difference between these dimensions and the
assemblies’ distances extracted from FFT measurements on
clustered aggregates from 3 and 4 (vide supra) indicates that
their hydrophilic domain is not visible in TEM, which is a known
phenomenon in the case of PEG head groups.42 In the case of
aggregates from 1 and 3 (C18), the articulate electron density
disparities between the bi-aromatic central spacer and the
hydrocarbon tails generate a density profile (light centre and
dark edges) for the hydrophobic region alone, which clearly
suggest
a
bilayer (1) or bimolecular (3) arrangement,
respectively.
Besides this straight forward construction model, there also
remain some observations to be mentioned. The observed 8.5
nm bilayer thickness (1) or diameter of the rods (3) are both
larger than expected from the molecular structure of the
constituting amphiphiles, i.e. they are larger than double the
molecules hydrophobic length (vide supra). On the other hand,
visible diameters of the micellar threads and globular micelles
from compounds 2 and 4, respectively, are slightly smaller (5
instead of 6 nm) in this regard. Also note that, although
amphiphiles 3 and 4 contain the same mPEG1000 head groups,
the calculated hydrophilic shell thickness (Table 1) of the
micelles from 4 (1.6 nm) is about 38% thinner than that of the
micellar rods from 3 (2.6 nm). While both these short values
clearly point to coiled arrangements of the PEG chains, beyond
this, one can only speculate about the origin of the marked shell
thickness difference. It might be caused by slightly differing
packings - most probably due to the bi-aromatic spacers - of the
short and long tailed twinned amphiphiles. The thinner
hydrophilic shell hints to a more loose packing within the small
micelles, which provides more lateral space for the PEG chains
to coil resulting in a shorter radial extent. To the contrary, a
dense packing of the hydrophobic cores forces the PEG chains
to extend further and thus, effects a thicker hydrophilic shell. A
tight assembly would also explain the slightly oversized visible
thickness of the ribbons from 1 and the diameters of the rods
Figure 7. Aggregation behaviour of twinned amphiphiles 1-4. Amphiphile 1
containing a comparatively large hydrophobic part and only short hydrophilic
PEO chains resembles
a cylindrical shape and hence forms ribbon-like
aggregates, i.e. planar bilayers (top row). The long PEO chains and short alkyl
moieties result in a prominently cone-shaped geometry in the case of 4 which
assemble into highly curved globular micelles (bottom row). In the case of 2
and 3, where head groups are only moderately larger than the hydrophobic
tails, and slightly cone-shaped geometry drives the aggregation towards
fibrous or rod-like nanostructures (middle row).
Table 1. Structural data of amphiphiles 1 – 4.
Compound
Aggreg
ate type
Visible
thickness/dia
meter
Overall
diameter
*
Calculated
invisible
shell*
[
nm]
[nm]
[nm]
C
18/mPEG350
11/mPEG350
18/mPEG1000
11/mPEG1000
ribbons
8.5 ± 0.5
-
-
-
(1)
C
micellar
threads
micellar
rods
globular
micelles
5.1 ± 0.4
8.5 ± 0.5
5.0 ± 1.0
-
(2)
C
13.8 ±
0.3
2.6 ± 0.4
1.6 ± 0.5
(3)
C
8.2 ± 0.3
(4)
*
Diameters calculated from FFT of densely arranged aggregates which were
found only in preparations of 3 and 4.
Table 1 clearly demonstrates coinciding dimensionalities of the
aggregates with regard to the lengths of the amphiphiles. Thus,
aggregates from both compounds containing the stearic acid
tails (1 and 3) have a visible thickness/diameter of about 8.5 nm.
Whereas the aggregates from undecenoic acid substituted
amphiphiles (2 and 4) display diameters of about 5 nm.
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from 3. Here, one may assume that the innermost parts of the
PEG chains might contribute some contrast to the TEM
micrographs due to a compacted arrangement next to the
hydrophobic centres or that the core region is not exactly
determined due to packing offsets like those that were shown
earlier by Padia et al. for short PEO chain amphiphiles.[43]
Likewise, the varying stiffness of the micellar threads (2) and the
rods (3) hints to more compact packing within the latter.
Nevertheless, since we lack a measure for the shell thickness of
the aggregates from 1 and 2 this explanation remains partly
hypothetic. Finally, although no direct evidence like a bilayer
profile was found for the micellar threads and globular micelles
from 2 and 4, respectively, it seems to be reasonable to also
assume a bimolecular construction with the PEG shell being
invisible, again.
Guest encapsulation study
Our interest is to understand the influence of different structural
constituents of these twinned amphiphiles on their encapsulation
capacity. The potential of these amphiphiles as nanocarriers for
hydrophobic guests was investigated using two hydrophobic
guest molecules i.e. Nile Red and Nimodipine. Nile Red is a
poorly water soluble, neutral and environmentally sensitive
fluorescent dye. It exhibit highly solvatochromic fluorescence
with strong emission in lipophilic environment.44 Nile red
encapsulation gives fair amount of information about the site of
encapsulation in nano-architectures. While Nimodipine (NIM), a
derivative of 1,4-dihydropyridine developed by Bayer AG in
1983, is a well-known calcium channel blocker. It is used to
increase the cerebral blood flow in humans as well as in
4
5
-
a
n
i
m
a
l
s
.
H
o
w
e
v
e
r
,
d
u
e
t
o
i
t
s
p
o
o
r
a
q
u
e
o
u
s
s
o
l
u
b
i
l
i
t
y
(
0
.
4
m
g
/
L
1)
, the commercial formulation involves the use of polyethylene
Cytotoxicity
glycol (62.5 g) and ethanol (37.5 g) to solubilize 50 mg of
Nimodipine.34 Since our amphiphiles are constituted from PEG,
To test the suitability of the amphiphiles 2, 3, and 4 for biological
and clinical applications, a cytotoxicity study was performed by
real time cell analysis (RTCA) using human A549 lung cancer
cells (Figure 8). All amphiphiles were well tolerated up to a
concentration of 0.1 mg ml-1 and even an extended incubation
for up to 3 days did not result in an increased toxicity (SI Figure
they can enhance the solubility of Nimodipine besides facilitating
its encapsulation and circulation in the biological systems.
The guest encapsulation experiments were carried out as
reported in the literature.46
A detailed description of the
procedure as well as the mode of calculation of the
encapsulation capacity is provided in the experimental
section/ESI. Amphiphile 1 could not be considered for studying
the transport potential due to its poor solubility in water. The
transport capacity of different amphiphiles with identical mPEG
and alkyl chains showed similar trends for the two different guest
20). Amphiphile 3 with the longest alkyl chain showed the best
compatibility and was even non-toxic up to the highest test
concentration of 1 mg ml-1 and hence is considered most
suitable for later medicinal applications e.g. as nano carrier for
drug encapsulation.
molecules, indicating
a strong relation between transport
capacity and structure of the hosting amphiphile. The transport
capacity of Nile Red is found to be in line with our previous
report on nonionic dendritic amphiphiles displaying unexpected
parameters in micellar assemblies.21 While for the Nimodipinie
these systems found to be better than our previous reported
system.34,46,47 For amphiphiles 2 and 4 having short C11 alkyl
chain, it was observed that increasing the length of the mPEG
head group from Mn 350 (2) to 1000 (4) leads to considerably
decreased transport capacity for either of the guest molecules.
However, increasing the length of the alkyl chain from C11 to C18
like in case of amphiphile 3 led to an enhancement of the
transport capacity (Figure 9 and Table 2). Qualitative analysis
of the data shows that the transport capacity depends on the
length of both alkyl and mPEG chains, and hydrophilic-lipophilic
balance of the amphiphile.
Figure 8: Cell viability test of amphiphiles 2, 3, and 4 by real time cell analysis
(RTCA) of A549 adenocarcinomic human alveolar basal epithelial cells. End
point data of cells treated for 24 hours was used to calculate the toxicity of the
amphiphiles compared to non-treated control cells. The cytostatic drug
doxorubicin (1 µM) was used as a positive control. Measurements were
performed in triplicate and data represent the mean ± standard deviation (SD).
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loading capacity and HLB of the amphiphiles emerged. The
mPEG based twinned amphiphilic architecture introduced herein
was found to be more efficient in means of drug loading capacity
as compared to the dendritic amphiphilic architecture which was
earlier reported by Thota et al.46 The combined synthetic
approach of simple and environmentally safe chemical steps
along with the understanding of the aggregation behaviour
observed for the synthesized amphiphiles opens up possibilities
for design, synthesis and applications of new structural motifs
based on twinned PEG amphiphiles.
1
10
00
1
Nile Red
Nimodipine
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
Experimental Section
2
3
4
Amphiphiles
All the compounds were characterized by their physical and
spectral data. Infrared spectra were recorded on a Perkin-Elmer
FT-IR model 9 spectrophotometer. The 1H and 13C NMR spectra
Figure 9. Evaluation of transport capacity of the amphiphiles 2, 3, and 4 using
Nile red and Nimodipine.
recorded on Jeol-400 (400 MHz, 100.5 MHz) NMR spectrometer
using TMS as an internal standard. The chemical shift values are
measured on δ scale and the coupling constant values (J) are
reported in Hz. The HRMS data were recorded on Agilent-6530,
Q-TOF LCMS.
Table 2. Calculation of transport capacity in mg/g [dye or drug (mg)
/
amphiphiles (g)].
Compound
Transport capacity (mg/g)
Nile Red
10.18
8.03
Nimodipine
83.04
C
C
C
11/mPEG350 (2)
18/mPEG1000 (3)
11/mPEG1000 (4)
96.09
All of the chemicals and solvents used were purchased from the
Spectrochem Pvt. Ltd., SD Fine Chemicals Pvt. Ltd., Sigma-
Aldrich, and Alfa Aesar. All the solvents were distilled prior to
their use. Novozym 435 (immobilized Candida antarctica lipase)
was purchased from Julich Chiral Solutions GmbH (Jülich,
Germany). Reactions were monitored by pre-coated TLC plates
7.23
69.74
Conclusions
A
new series of non-ionic twinned amphiphiles with two
hydrophilic mPEG head groups and two hydrophobic alkyl
chains has been synthesized. The non-ionic PEG based
amphiphiles have been readily prepared by utilizing ‘Click
chemistry’ and acylation reactions on a chemo-enzymatically
(
Merck silica gel 60 F254), by visualizing the spot in ceric
solution stain and iodine. All the compounds were purified by
column chromatography using silica gel (100-200 mesh).
Procedure for the synthesis of 2-azidopropane-1,3-diol
2 2
synthesized A B monomer. The aggregation behaviour of these
amphiphiles was investigated using fluorescence and DLS.
Complementing on cryo-TEM measurements which proved the
formation of globular, thread-like or rod-like micelles as well as
planar double-layer assemblies, depending on the amphiphile’s
molecular structure. The observed morphologies are in line with
the packing parameter model of Israelachvili. Homogeneous
ordering of the aggregates from amphiphiles 3 and 4 at elevated
concentrations provided the additional opportunity for
determining the structural dimensions with high accuracy. The
cytotoxicity study suggested the twinned amphiphiles 2-4 to be
suitable for in vivo applications, their potential as nanocarriers
was investigated using hydrophobic guest molecules Nile Red
and Nimodipine showing that the Nile Red was more efficiently
encapsulated by amphiphiles having lower HLB value if
compared to Nimodipine. Additionally, a correlation between
1
,3-Diacetoxyglycerol
and
2-azidopropane-1,3-diol
were
synthesized according to the published procedures and spectral
data obtained were found to be identical with those reported in
the literature.36
2-Azidopropane-1,3-diyl distearate (5)
2-Azidopropane-1,3-diol (1 g, 8.54 mmol) and stearic acid (7.29
g, 25.64 mmol) were dissolved in DCM (30 mL). The solution
o
was maintained at 0 C, then EDC.HCl (4.89 g, 25.64 mmol) and
DMAP (1.25 g, 10.25 mmol) were added. The reaction mixture
was allowed to warm up to room temperature and stirred at 30
oC for 15 h. Progress of the reaction was monitored by TLC
using ethyl actate/petroleum ether (1:9) (R
f
= 0.6). On
completion of the reaction, the reaction mixture was
concentrated under reduced pressure and the obtained crude
was suspended in water and ethyl acetate (3 x 30 mL). The
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combined organic layers were dried over anhydrous Na
2
SO
4
and
The reaction mixture was allowed to warm at room temperature
and left for stirring at 80 oC for 24 h. Progress of the reaction
was monitored by TLC. On completion of the reaction, the black
residue was filtered off. The filtrate was acidified using 2 M
hydrochloric acid solution to pH 2 and then extracted with
the solvent evaporated to yield the crude product, which was
purified through column chromatography using ethyl
acetate/petroleum ether to give the desired compound 5 in 93%
yield. 1H NMR (400 MHz, CDCl
OCH(CH O-) ), 3.47-3.45 (m, 1H, -N
H, -OOCCH CH -), 1.67-1.59 (m, 4H, -OOCCH
.20 (m, 56H, Alkyl chain), 0.88 ppm (t, J = 4.0 Hz, 6H, -CH
): δ = 173.41, 63.15, 58.83, 34.15,
2.07, 29.83, 29.59, 29.38, 29.24, 24.95, 22.83, 14.25. HRMS:
m/z [M+Na+] calcd for
649.5758; found:
): δ = 4.31-4.11 (m, 4H, -
2
CH(CH O-) ), 2.37-2.29 (m, chloroform (4 x 50 mL). The organic layers was dried over
3
2
2
3
2
4
1
2
2
2
CH
2
-), 1.29-
anhydrous sodium sulfate, and concentrated under reduced
1
3
).
pressure to get the desired mPEG acid, 7 in 91% yield; H NMR
13C NMR (100.5 MHz, CDCl
(400 MHz, CDCl
3.50 (m, 24H, mPEG region), 3.34 (s, 3H, CH
NMR (100.5MHz, CDCl ): δ = 172.21, 71.80, 71.66, 70.33, 68.89,
): δ = 4.12 (s, 2H, HOOCCH
O-mPEG), 3.71-
3
3
2
3
3
O-mPEG). 13C
39
C H75NaN
3
O
4
:
3
-1
58.93. IR(neat) νmax = 2868, 1741, 1455 cm-1.
6
2
2
72.5660. IR(KBr) νmax 2916, 2849, 2130, 1743, 1467 cm .
-azidopropane-1,3-diyl bis(undec-10-enoate) (6)
-Azidopropane-1,3-diol (1 g, 8.54 mmol) and undecylenic acid
mPEG-1000 acid (8)
w
Mono-methoxy poly(ethylene glycol) (M : 1000) (5 g, 10 mmol)
(
7.29 g, 25.64 mmol) were dissolved in DCM (30 mL). The
was dissolved in distilled water (50 mL) in a 250 mL round-
bottom flask and 0.8 g, 40 mmol of sodium hydroxide was added.
The reaction mixture was stirred in ice bath to allow the
solution was kept on ice to maintain the temperature at 0 oC,
then EDC.HCl (1.25 g, 10.25 mmol) and DMAP (4.89 g, 25.6
mmol) were added. The reaction mixture was allowed to warm
o
temperature to reach 0 C, then (4.7 g, 60 mmol) of potassium
o
up to room temperature and stirred at 30 C for 15 h. Progress of
permanganate was added in small amounts for approx. 1-2 hr.
The reaction mixture was allowed to warm at room temperature
the reaction was monitored by TLC using ethyl actate/petroleum
o
ether (1:9) (R
f
= 0.6). Upon completion of the reaction, the
and then heated to 80 C for 24 h. Progress of the reaction was
reaction mixture was concentrated under reduced pressure and
obtained product was extracted with ethyl acetate (3 x 30 mL)
and water. The combined organic layers were dried over
monitored by TLC. On completion of the reaction, black residue
was filtered off. The filtrate was acidified using 2 M hydrochloric
acid solution to pH 2 and then extracted with chloroform (6 x 50
mL). The organic layers was dried over anhydrous sodium
2 4
anhydrous Na SO and the solvent evaporated to yield the crude
product, which was then purified through column
sulfate, and concentrated under reduced pressure to get the
1
chromatography using ethyl acetate/petroleum ether to give the
desired mPEG acid, 8 in 91% yield. H NMR (400 MHz, CDCl
3
):
1
desired compound 6 in 93% yield. H NMR (400 MHz, CDCl
3
): δ
=CH-),
), 3.89-3.83 (m, 1H,
), 2.36-2.29 (m, 4H, -OOCCH CH -), 2.05-2.00 (m,
=CH-CH -), 1.64-1.60 (m, 4H, -OOCCH CH -), 1.40-
.25 (m, 20H, alkyl chain). 13C NMR (100.5 MHz, CDCl
): δ =
δ = 4.10 (s, 2H, HOOCCH
region), 3.37 (s, 3H, CH
3
CDCl ): δ = 172.0, 71.88, 70.95, 70.51, 68.80, 58.98. IR (Film)
2
O-mPEG), 3.70-3.53 (m, 83H, mPEG,
=
5.84-5.74 (m, 2H, CH
.24-4.10 (m, 4H, -OCH(CH
CH(CH O-)
H, CH
2
=CH-), 5.00-4.90 (m, 4H, CH
2
3
O-mPEG). 13C NMR (100.5 MHz,
4
2
O-)
2
-
-1
N
3
2
2
2
2
ν
max = 2865, 1743, 1465 cm .
4
1
1
2
2
2
2
2
2-(4-(ethoxycarbonyl)phenoxy)propane-1,3-diyl diacetate (9)
3
To a stirring solution of ethyl 4-hydroxybenzoate (1 g, 6.02
73.33, 139.24, 114.26, 63.11, 58.79, 34.09, 33.87, 29.36, 29.20, mmol), 1, 3-diacetoxyglycerol (1.5 g, 9.02 mmol) and
+
9.16, 29.13, 28.98, 24.89. HRMS: m/z [M+Na ] calcd for
triphenylphosphine (3.15 g, 12.03 mmol) in toluene (20 mL),
diethyl azodicarboxylate in 5 mL of toluene was added drop wise.
C
25
H43NaN
3
O
4
: 449.3254; found: 472.3174. IR (Film) νmax
=
-1
o
2
952, 2854, 2125, 1740, 1462 cm .
The reaction mixture was stirred for 15 h at 30 C. The progress
mPEG-350 acid (7)
of the reaction was monitored by TLC using ethyl
Mono-methoxy poly(ethylene glycol) (M
w
: 350) (5 g, 10 mmol)
f
.
acetate/petroleum ether (3:7) (R = 0.4) On completion of the
was dissolved in distilled water (50 mL) in a 250 mL round-
bottom flask and (2.2 g, 40 mmol) of sodium hydroxide was
added. The reaction mixture was stirred in ice bath to allow the
reaction, toluene was removed under reduced pressure and the
crude was suspended in water and ethyl acetate (3 x 30 mL).
2 4
The combined organic layers were dried over Na SO and the
o
temperature to reach 0 C, then 13.5 g, 60 mmol of potassium
solvent evaporated to yield the crude product, which was
purified through column chromatography using petroleum
permanganate was added in small amounts for approx. 2 to 3 hr.
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ether/ethyl acetate to give the desired compound 9 as a viscous
(m, 4H, -OCH(CH
13C NMR (100.5 MHz, CDCl
122.62, 115.51, 78.33, 77.98, 75.06, 62.05, 52.44. HRMS : m/z
[M+Na+ ] calcd for C13
: 212.0685; found: 235.0586.
2
OH)
2
), 2.50 (t, J = 2.3 Hz, 1H, -CH
2
C≡CH).
1
liquid in 83% yield. H NMR (400 MHz, CDCl
3
): δ = 7.99 (d, J =
3
) : δ = 165.51, 161.99, 132.19,
8.0 Hz, 2H, aromatic CH), 6.99 (d, J = 8.0 Hz, 2H, aromatic CH),
4
.79-4.75 (m, 1H, -OCH(CH
O-) ) and -OCH CH ), 2.06 (s, 6H, -COCH
8.0 Hz, 3H, -CH CH
2
O-)
2
), 4.37-4.26 (m, 6H,
-
H14NaO
5
-1
OCH(CH
2
2
2
3
3
), 1.37 (t, J
IR(neat) νmax = 3291, 2309, 1713, 1604, 1508 cm .
=
2
3
). 13C NMR (100.5 MHz, CDCl
3
): δ =
Synthesis of compound 12
1
70.79, 166.29, 161.41, 131.81, 124.15, 115.45, 73.57, 62.75,
Compound 11 (0.5 g, 2 mmol) and 2-azidopropane-1,3-diyl
distearate (5, 1.56 g, 2.4 mmol) were dissolved in 20 mL
THF/water (3:1) followed by the addition of copper sulfate
(0.199 g, 0.8 mmol) and sodium ascorbate (0.079 g, 0.4 mmol).
+
60.91, 20.87, 14.50. HRMS (positive, MeOH): m/z [M+Na ]:
7
calcd for C16H20NaO : 324.1209; found: 347.1089. IR(film) νmax =
-1
3743, 2981, 1743, 1710, 1605, 1510 cm .
o
4-((1,3-dihydroxypropan-2-yl)oxy)benzoic acid (10)
The reaction mixture was then stirred at 50 C for 15 h. Progress
Compound 9 (6 g, 15.43 mmol) was dissolved in a methanolic
of the reaction was monitored by TLC using chloroform/
solution of NaOH (1N) (30 mL) and water (10 mL), the reaction
f
methanol (9:1) (R = 0.55). On completion of the reaction, the
o
mixture was stirred at 50 C for 4 h. Progress of the reaction was
reaction mixture was concentrated under reduced pressure and
the obtained crude product was suspended in chloroform (3 x 20
mL) and water. The combined organic layers were dried over
f
monitored by TLC using methanol/chloroform (1:9) (R = 0.15).
Upon the completion of the reaction, reaction mixture was
neutralized with dil. HCl and concentrated under reduced
pressure to obtain the crude product which was purified through
column chromatography using chloroform/methanol to give the
desired compound 10 in 95% yield. 1H NMR (400 MHz,
2 4
anhydrous Na SO and the solvent evaporated to yield the crude
product which was subsequently purified through column
chromatography using chloroform/methanol to give the desired
1
compound 12 in 93% yield. H NMR (400 MHz, CDCl
3
): δ = 7.95
CD
3
OD): 7.97 (d, J = 8.0 Hz, 2H, aromatic CH), 7.08 (d, J = 8.0
OH) ), 3.81-
). 13C NMR (100.5 MHz, CD
OD): δ
(d, J = 8.0 Hz, 2H, aromatic CH), 7.78 (s, 1H, triazolyl ring CH),
6.95 (d, J = 8.0 Hz, 2H, aromatic CH), 5.42 (s, 2H, -OCH
triazole), 5.10-5.04 (m, 1H, triazole-CH(CH O-) ), 4.52-4.48 (m,
5H, -OCH(CH OH) and triazole-CH(CH O-) ), 3.95-3.86 (m, 4H,
-OCH(CH OH) ), 2.34-2.24 (m, 2H, -OOCCH CH -), 1.58-1.51
(m, 4H, -OOCCH CH -), 1.28-1.23 (m, 56H, alkyl chain), 0.87 (t,
J = 4.0 Hz, 6H, -CH ): δ = 172.83,
) ). 13C NMR (100.5 MHz,CDCl
Hz, 2H, aromatic CH), 4.87-4.49 (m, 1H, -OCH(CH
2
2
2
-
3.78 (m, 4H, -OCH(CH
2
OH)
2
3
2
2
=
169.90, 164.02, 132.81, 124.18, 116.60, 80.43, 61.87. HRMS:
2
2
2
2
+
m/z [M+Na ] calcd for C10
H12NaO
5
: 212.0685; found: 235.0586.
2
2
2
2
-1
IR(neat) νmax = 3736, 3615, 2925, 2853, 2312, 1740, 1514 cm .
Prop-2-yn-1-yl 4-((1,3-dihydroxypropan-2-yl)oxy)benzoate
11)
2
2
3
3
(
165.82, 161.66, 142.95, 131.76, 123.62, 122.54, 115.16, 78.09,
62.04, 61.70, 58.49, 57.52, 33.67, 31.73, 29.51, 29.46, 29.41,
To a solution of compound 10 (3.5 g, 16.50 mmol) in DMF (40
mL), potassium carbonate (4.5 g, 33 mmol) was added, the
29.25, 29.16, 29.01, 28.84, 24.51, 22.49, 13.92. HRMS: m/z
o
+
reaction mixture was stirred for 15 min at 60 C. To this reaction
[M+Na ] calcd for C52
H89NaN
3
O
9
: 899.6599; found: 922.6496. IR
-1
mixture, propargyl bromide was slowly added (2.35 g, 19.80
(Film) νmax = 3743, 2914, 2855, 1738, 1463 cm .
o
mmol). This reaction mixture stirred at 60 C for 6 h. Progress of
Synthesis of compound 13
the reaction was monitored by TLC using chloroform/methanol
Compound 11 (0.5 g, 2 mmol) and 2-azidopropane-1,3-diyl
bis(undec-10-enoate) (6, 1.08 g, 2.4 mmol) were dissolved in 20
mL THF/water (3:1) followed by the addition of copper sulfate
(0.199 g, 0.8 mmol) and sodium ascorbate (0.079 g, 0.4 mmol).
(
f
9:1) (R = 0.3). On completion of the reaction, reaction mixture
was concentrated under reduced pressure and the obtained
crude product was suspended in water and ethyl acetate (3 x 25
o
mL). The combined organic layers were dried over Na
2
SO
4
and
The reaction mixture was then stirred at 50 C for 15 h. Progress
the solvent evaporated to yield the crude product, which was
purified through column chromatography using chloroform
of the reaction was monitored by TLC using chloroform/
f
methanol (9:1) (R = 0.55). On completion of the reaction, the
/
methanol to give the desired compound 11 in 87% yield. 1
NMR (400 MHz, CDCl ): δ = 7.98 (d, J = 8.0 Hz, 2H, aromatic
CH), 6.98 (d, J = 8.0 Hz, 2H, aromatic CH), 4.86 (d, J = 2.3 Hz,
H, -CH C≡CH), 4.54-4.49 (m, 1H, -OCH(CH OH) ), 3.94-3.86
H
reaction mixture was concentrated under reduced pressure and
the obtained crude product was suspended in water and
chloroform (3 x 20 mL). The combined organic layers were dried
3
2
2
2
2
2 4
over anhydrous Na SO . The solvent evaporated to yield the
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crude product, which was subsequently purified through column
22.83, 14.26. GPC: Mw = 1979, Mn = 1918, PDI = 1.032; IR
-1
chromatography using chloroform/methanol to give the desired
(neat) νmax = 2915, 2851, 1735, 1605, 1508 cm .
1
compound 13 in 93% yield. H NMR (400 MHz, CDCl
3
): δ = 7.96
Synthesis of compound 2
(
d, J = 8.0 Hz, 2H, aromatic CH), 7.78 (s, 1H, triazolyl ring CH),
Compound 13 (7, 0.5 g, 0.71 mmol) and mPeg 350 acid (0.751 g,
2.14 mmol) were dissolved in DCM (30 mL). The reaction
mixture was stirred under ice cold conditions to maintain the
6
.96 (d, J = 8.0 Hz, 2H, aromatic CH), 5.84-5.74 (m, 2H,
=CH-), 5.43 (s, 2H, -OCH -triazole), 5.11-5.05 (m, 1H,
triazole-CH(CH O-) ), 5.00-4.90 (m, 4H, CH
H, -OCH(CH OH) ) and triazole-CH(CH O-)
), 2.28-2.24 (m, 4H, -OOCCH
=CH-CH -), 1.62-1.52 (m, 4H, -OOCCH
CH
2
2
=CH-), 4.55-4.46 (m. temperature at 0 oC, then EDC.HCl (0.318 g, 1.66 mmol) and
2
2
2
5
2
2
2
2
), 3.96-3.88 (m, 4H, DMAP (0.081 g, 0.66 mmol) were added. The reaction mixture
-
(
OCH(CH
2
OH)
2
2
CH
2
-), 2.04-1.99
CH -), 1.37-
): δ =
was allowed to warm at room temperature and left stirring at 30
oC for 15 h. Progress of the reaction was monitored by TLC
m, 4H, CH
2
2
2
2
1
1
1
2
.18(m, 20 H, alkyl chain). 13C NMR (100.5 MHz, CDCl
3
f
using chloroform/methanol (9:1) (R = 0.4). Upon completion of
72.79, 165.76, 161.67, 142.93, 138.94, 131.73, 123.61, 122.47,
15.14, 113.97, 62.03, 61.66, 58.49, 57.50, 33.64, 29.03, 28.98,
the reaction, reaction mixture was concentrated under reduced
pressure and obtained crude product was suspended in water
and chloroform (3 x 30 mL). The combined organic layers were
8.82, 28.65, 24.47. HRMS: m/z [M+Na+
: 699.4095; found: 722.4019. IR (neat) νmax
]
calcd for
C
38
H57NaN
3
O
9
=
2 4
dried over anhydrous Na SO and the solvent evaporated to
-1
2
923, 2864, 1742, 1604, 1508 cm .
yield the crude product which was purified through column
Synthesis of compound 1
chromatography using chloroform/methanol to yield the desired
1
Compound 12 (0.5 g, 0.55 mmol) and mPEG 350 acid (7, 0.583
g, 1.66 mmol) were dissolved in DCM (30 mL). The mixture was
stirred under ice cold conditions to maintain the temperature at
0oC. Then EDC.HCl (0.318 g, 1.66 mmol) and DMAP (0.081 g,
compound 2 in 91% yield. H NMR (400 MHz, CDCl
3
): δ = 7.98
(d, J = 8.0, 2H, aromatic CH), 7.77 (s, 1H, triazolyl ring CH), 6.97
(d, J = 8.0 Hz, 2H, aromatic CH), 5.82-5.76 (m, 2H, CH =CH-),
5.44 (s, 2H, -OCH -triazole), 5.08-5.06 (m, 1H, triazole-
), 4.99-4.90 (m, 4H, CH =CH-), 4.80-4.77 (m, 1H, -
), 4.54-4.49 (m, 4H, triazole-CH(CH O-) ), 4.43-4.34
O-) ), 4.15 (s, 4H, -OOCCH O-mPEG), 3.73-
3.53 (m, 60H, mPEG region), 3.37 (s, 6H, CH O-mPEG), 2.27 (t,
J = 4.0 Hz, 4H, -OOCCH CH -), 2.03-2.00 (m, 4H, CH =CH-
-), 1.55-153 (m, 4H, -OOCCH CH -), 1.37-1.33 (m, 4H, -
-), 1.29-1.25 (m, 16H, alkyl chain). 13C NMR (100.5 MHz,
): δ = 172.95, 170.89, 170.12, 165.81, 161.32, 139.12,
2
2
0.66 mmol) were added. The reaction mixture was allowed to
CH(CH
2
2
O-)
2
2
2
o
warm up to room temperature and left for stirring at 30 C for 15
h. Progress of the reaction was monitored by TLC using
CH(CH
O-)
2
2
(m, 4H, -CH(CH
2
2
2
chloroform/methanol (9:1) (R
f
= 0.4). On completion of the
3
reaction, the reaction mixture was concentrated under reduced
pressure and the obtained crude was suspended in water and
chloroform (3 x 20 mL). The combined organic layers were dried
2
2
2
CH
2
2
2
2
CH
over anhydrous Na
2
SO
4
and the solvent evaporated to yield the
CDCl
3
crude product which was further purified through column
132.01, 123.75, 123.32, 115.31, 114.18, 73.12, 71.93, 70.67,
68.35, 62.47, 62.25, 59.02, 58.66, 57.82, 33.86, 33.75, 29.23,
29.02, 28.98, 28.85, 24.67 ppm; GPC: Mw = 1513, Mn = 1462,
PDI = 1.034. IR (Film) νmax = 2962, 2883, 1744, 1605, 1508,
chromatography using chloroform/methanol to give the desired
1
compound 1 in 91% yield. H NMR (400 MHz, CDCl
3
): δ = 7.99
(
d, J = 8.0 Hz, 2H, aromatic CH), 7.77 (s, 1H, triazolyl ring CH),
-
1
6
.98 (d, J = 8.0 Hz, 2H, aromatic CH), 5.44 (s, 2H, -OCH
O-) ), 4.80-4.78 (m,
), 4.55-4.49 (m, 4H, triazole-CH(CH O-) ),
.43-4.34 (m, 4H, -CH(CH O-) ), 4.16 (s, 4H, -OOCCH
2
-
1454 cm .
triazole), 5.09-5.06 (m, 1H, triazole-CH(CH
2
2
Synthesis of compound 3
1H, -OCH(CH
2
O-)
2
2
2
Compound 12 (0.5 g, 0.55 mmol) and mPEG 1000 acid (1.66 g,
1.66 mmol) were dissolved in DCM (30 mL). The reaction
mixture was stirred under ice cold conditions to maintain the
temperature at 0 oC, then EDC.HCl (0.409 g, 2.14 mmol) and
DMAP (0.104 g, 0.85 mmol) were added. After that, the reaction
mixture was allow to warm at room temperature and left for
4
2
2
2
3
O-
O-
mPEG), 3.75-3.54 (m, 44H, mPEG region), 3.37 (s, 6H, CH
PEG), 2.28 (t, J = 4.0 Hz, 4H, -OOCCH CH
OOCCH CH -) 1.30-1.24 (m, 56H, alkyl chain), 0.87 (t, J = 4.0
Hz, 6H, -CH ): δ = 173.11,
). 13C NMR (100.5 MHz, CDCl
70.27, 165.96, 161.46, 143.23, 132.17, 123.90, 123.46, 115.46,
2
2
-), 1.54-1.56 (m, 4H,
-
2
2
3
3
o
1
7
3
stirring at 30 C for 15 h. Progress of the reaction was monitored
2.08, 71.16, 70.73, 68.51, 62.64, 62.39, 59.17, 58.81, 57.95,
4.02, 32.06, 29.84, 29.80, 29.74, 29.49, 29.35, 29.18, 24.86,
by TLC using chloroform/methanol (9:1) (R
f
= 0.4). Upon
completion of the reaction, the reaction mixture was
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Chemistry - An Asian Journal
10.1002/asia.201700450
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concentrated under reduced pressure and the obtained crude
was suspended in water and chloroform (3 x 20 mL). The
4.0 Hz, 4H, -OOCCH
1.50-1.47 (m, 4H, -OOCCH
chain). 13C NMR (100.5 MHz, CDCl
3
2
CH
2
-)1.99-1.96 (m, 4H, -CH
CH -), 1.34-1.19 (m, 20H, alkyl
): δ = 173.09, 170.23,
2
2
=CH-CH -),
2
2
combined organic layers were dried over anhydrous Na
the solvent evaporated to yield the crude product which was
purified through column chromatography using
chloroform/methanol to give the desired compound 3 in 91%
yield. 1H NMR (400 MHz, CDCl
): δ 7.97 (d, J = 8.0 Hz, 2H,
aromatic CH), 7.77 (s, 1H, triazolyl ring CH), 6.98 (d, J = 8.0 Hz,
H, aromatic CH), 5.44 (s, 2H, -OCH -triazole), 5.09-5.06 (m, 1H,
triazole-CH(CH O-) ), 4.80-4.77 (m, 1H, -CH(CH O-) ), 4.54-
.47 (m, 4H, triazole-CH(CH O-) ), 4.43-4.29 (m, 4H,
CH(CH O-) ), 4.17-4.15 (m, 4H, -OOCCH O-mPEG), 3.74-3.53
m, 190H, mPEG region), 3.37 (s, 6H, CH O-mPEG), 2.27 (t, J =
2
SO
4
and
166.15, 161.25, 143.19, 139.25, 132.12, 123.89, 115.38, 114.30,
72.02, 70.64, 70.64, 68.40, 62.55, 62.34, 59.14, 58.73, 33.96,
33.87, 29.34, 29.23, 29.13, 29.08, 28.95, 24.78. GPC: Mw =
2339, Mn = 1967, PDI = 1.189, IR (neat) νmax = 2914, 2855, 1737,
3
-1
1606, 1463 cm .
2
2
Acknowledgements
2
2
2
2
We gratefully acknowledge the financial support from German
Science Foundation (DFG) and SERB-DST, Government of
India, and University of Delhi.
4
2
2
-
2
2
2
(
3
Keywords: Twinned amphiphiles • Self-assembly• cryo-TEM •
nanocarriers • Nile Red, Nimodipine
4
.0 Hz, 4H, -OOCCH
2
CH
2
-), 1.56-1.53 (m, 4H, -OOCCH
2
CH
2
-),
).
1.29-1.24 (m, 56H, alkyl chain), 0.87 (t, J = 4.0 Hz, 6H, -CH
3
13C NMR (100 MHz, CDCl
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2
32.03, 124.02, 123.97, 115.47, 109.96, 73.08, 71.89, 70.52,
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[
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1% yileld. 1H NMR (400 MHz, CDCl
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Chemistry - An Asian Journal
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FULL PAPER
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:
Entry for the Table of Contents
A new class of twinned amphiphiles
was developed by utilizing ‘Click
chemistry’ and acylation reactions on
a
chemo-enzymatically synthesized
2 2
B monomer, the aggregation
A
behaviour of these amphiphiles was
investigated using fluorescence, DLS,
cryo-TEM studies. The cryo-TEM
measurements prove the formation of
globular, thread-like or rod-like
micelles as well as planar double-
layer assemblies, depending on the
amphiphile’s molecular structure. The
cytotoxicity study suggested the
twinned amphiphiles to be suitable for
in vivo applications. The transport
capacity results for both Nimodipine
and Nile Red prove them as
a
promising candidate for drug delivery.
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