Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies, and the Encapsulation and Release of Dyes through DNA-Mediated Disassembly

Article abstract of DOI:10.1002/chem.201502955

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di- and monoimide chromophores and the investigation of their photophysical, self-assembly and DNA-binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG.dC)-poly(dG.dC) and poly(dA.dT)-poly(dA.dT). The interaction of these conjugates with DNA was evaluated by photo- and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5-8×10⁴ M^-1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM, which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.

Full text of DOI:10.1002/chem.201502955

DOI: 10.1002/chem.201502955
Full Paper
&
Molecular Transport |Hot Paper|
Naphthalene Imide Conjugates: Formation of Supramolecular
Assemblies, and the Encapsulation and Release of Dyes through
DNA-Mediated Disassembly
[a]
[a]
[a, b]
Balaraman H. Shankar, Dhanya T. Jayaram, and Danaboyina Ramaiah*
Abstract: We report the synthesis of two new amphiphilic
conjugates 1 and 2 based on naphthalene di- and mono-
imide chromophores and the investigation of their photo-
physical, self-assembly and DNA-binding properties. These
conjugates showed aqueous good solubility and exhibited
strong interactions with DNA and polynucleotides such as
poly(dG·dC)–poly(dG·dC) and poly(dA·dT)–poly(dA·dT). The
interaction of these conjugates with DNA was evaluated by
photo- and biophysical techniques. These studies revealed
that the conjugates interact with DNA through intercalation
these two conjugates, bolaamphiphile 1 exhibited a supra-
molecular assembly that formed vesicles with an approxi-
mate diameter of 220 nm in the aqueous medium at a critical
aggregation concentration of 0.4 mm, which was confirmed
by SEM and TEM. These vesicular structures showed a strong
affinity for hydrophobic molecules such as Nile red through
encapsulation. Uniquely, when exposed to DNA the vesicles
disassembled, and therefore this transformation could be
utilised for the encapsulation and release of hydrophobic
molecules by employing DNA as a stimulus.
4
À1
with association constants in the order of 5–8”10 m . Of
Introduction
well-defined nanostructures because of their advantages
over conventional structures. In general, the stimulus em-
ployed to generate the desired nanoarchitecture can be
a change in temperature, pH, light, magnetic field or ionic
The self-assembly of amphiphilic conjugates has been an
active area of interest in recent years due to their medicinal
[1]
[5]
and material applications. Thus, small-molecule based amphi-
philes, such as surfactants, bolaamphiphiles and gemini surfac-
tants, have been employed to generate diverse nanostructures
strength of the medium. Among the various stimuli investi-
gated, materials that respond to biological stimuli, such as pro-
[
6]
teins and nucleic acids, have been less explored. Therefore,
the design of functional chromophores that form supramolec-
ular assemblies with biomolecule-responsive architectures in
aqueous media is quite challenging. Of the various chromo-
phores, systems based on naphthalene imides are very attrac-
tive biomolecule-responsive units because they have extended
p systems and belong to an important class of DNA-binding
[2]
(
e.g., micelles, vesicles, fibres and nanotubes). Most of these
amphiphiles contain hydrophobic and hydrophilic segments,
which are known to form various supramolecular assemblies in
[
3]
organic media through various non-covalent interactions.
However, creating well-defined nanostructures in aqueous
media for efficient biological applications is a great challenge.
In this context, supramolecular assemblies of appropriately
substituted chemical building blocks (such as amphiphilic and
[
7]
agents.
In this context, we have designed two amphiphilic conju-
gates 1 and 2 based on naphthalene imides and have investi-
gated their photophysical, self-assembly and DNA-responsive
properties. The uniqueness of these conjugates is their amphi-
philic nature, thus they are expected to exhibit good aqueous
solubility. The presence of the planar p-extended aromatic sur-
face in these systems makes them interesting candidates to ex-
plore the formation of bioresponsive supramolecular assem-
blies in aqueous media. Our investigations have revealed that
both conjugates 1 and 2 exhibited good solubility in the aque-
ous medium and had a strong affinity for DNA and polynucleo-
tides through intercalation. Of these two conjugates, bolaam-
phiphile 1 formed vesicular structures above the critical aggre-
gate concentration (CAC) of 0.4 mm, which could encapsulate
hydrophobic dye molecules. Interestingly, these supramolecu-
lar vesicular structures of conjugate 1 disassembled in the
presence of DNA, and this DNA-mediated transformation could
[
4]
bolaamphiphilic systems) have attracted much attention.
Recently, increasing efforts are being made towards the
development of stimuli-responsive units that contain
[
a] B. H. Shankar, Dr. D. T. Jayaram, Dr. D. Ramaiah
Photosciences and Photonics Section
Chemical Sciences and Technology Division
CSIR-National Institute for Interdisciplinary Science and
Technology (CSIR-NIIST), Trivandrum 695 019 (India)
E-mail: rama@niist.res.in
d.ramaiah@gmail.com
[
b] Dr. D. Ramaiah
CSIR-North East Institute of Science and Technology (CSIR-NEIST)
Jorhat 785 006, Assam (India)
E-mail: rama@rrljorhat.res.in
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502955. It contains detailed synthetic
procedures and characterisation data for compounds 1 and 2.
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be effectively utilised to encapsulate and release dyes in the
aqueous medium.
values for diimide conjugate 1 (F =0.005), relative to mono-
F
imide derivative 2 (F =0.29), which indicated an efficient in-
F
[
8]
tensity quenching for 1, as reported in the literature.
To determine the excited-state behaviour of conjugates
1
and 2 we carried out picosecond time-resolved fluorescence
measurements. The fluorescence lifetime (t), in the case of
conjugate 1, was found to be very short lived (t<0.1 ns),
which is a characteristic feature of the N-substituted NDI
[
9]
chromophore. The fluorescence decay profile of conjugate 2
lex =335 nm) resulted in a mono-exponential decay profile
(
Results and Discussion
with an emission lifetime of about 2.2Æ0.3 ns. This value can
be attributed to the local excited state (monomer) emission of
Synthesis
[
10]
the naphthalimide chromophore (Figure S1, see the Support-
ing Information).
Symmetric bolaamphiphile 1 was synthesised from naphtha-
lenetetracarboxylic dianhydride (NDA) in three steps
To understand the propensity of the conjugates under inves-
tigation to aggregate we monitored the absorption and fluor-
escence properties of 1 and 2 at higher concentrations.
Figure 2 shows the concentration-dependent absorption and
(
Scheme S1, see the Supporting Information). NDA reacted
with ethanolamine to afford the corresponding alcohol deriva-
tive, which was successively esterified with 11-bromoundeca-
noic acid (2 equiv) and quaternised with pyridine to give bo-
laamphiphile 1 in moderate yield (ꢀ66%). Similarly, the syn-
thesis of amphiphilic naphthalimide derivative 2 was achieved
in moderate yield (ꢀ55%) (Scheme S2, see the Supporting In-
formation) starting from naphthalic anhydride (NMA). Amina-
tion of NMA with ethanolamine, followed by esterification and
subsequent quaternisation with pyridine yielded conjugate 2.
All these starting materials and products were purified and un-
ambiguously characterised by various analytical and spectros-
copic techniques.
Photophysical and self-assembly properties
We investigated the photophysical properties, including time-
resolved fluorescence spectral analysis, of both conjugates
under different conditions. For example, the ground-state ab-
sorption spectrum of conjugate 1 in the aqueous medium
showed characteristic vibrationally resolved spectral bands of
the naphthalene diimide (NDI) chromophore (l =382 nm,
max
4
À1
À1
e=2.33Æ0.1”10 m cm ; Figure 1). For conjugate 2 we ob-
served the absorption maximum at l=344 nm (e=1.42Æ0.1”
4
À1
À1
1
0 m cm ). In the fluorescence spectra 1 and 2 showed
Figure 2. Concentration-dependent A) absorption and B) emission spectra
(normalised) of conjugate 1 in the aqueous medium. c=i) 46 mm to
vii) 1.1 mm. Inset: shows the variation of the extinction coefficient at
l=380 nm as a function of the concentration of conjugate 1. Path length of
the cell (l)=0.1 cm, lex =360 nm.
maxima at l=390 and 378 nm, respectively. The fluorescence
quantum yields (F ) of these conjugates were determined in
F
the aqueous medium. We observed significantly quenched
emission spectra of conjugate 1. As the concentration was in-
creased from 46 mm to 1.1 mm, we observed a deviation from
linearity at concentrations greater than about 0.4 mm. The CAC
of 1 was estimated from the change in the concentration-de-
pendent molar extinction coefficient (e) at l=382 nm to be
0
.4 mm (Figure 2A, inset). In the emission spectrum, we ob-
served a gradual increase in the emission at l=510 nm as the
concentration increased, with the I510/I390 ratio varying from 0.2
to 0.9. Similar observations were made for conjugate 2 (CAC
ꢀ
0.25 mm). In the emission studies, we observed the forma-
tion of a new band at l=500 nm. When monitored at l=
510 nm, conjugate 1 (at the CAC) decayed bi-exponentially
Figure 1. Absorption and emission (inset) spectra of conjugates 1 (10 mm;
c) and 2 (20 mm; d in the aqueous medium. lex =360 and 345 nm, re-
spectively.
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with an average fluorescence lifetime (<t>) of 3.3Æ0.2 ns,
which can be attributed to formation of the intermolecular ex-
cimer of the NDI (Figure S2, see the Supporting Information).
Similarly, conjugate 2 (at the CAC) showed a bi-exponential
decay with t=22.6Æ0.1 (90%) and 4.11Æ0.2 ns (10%), which
can be attributed to the excimer and monomer of the naph-
thalimide unit, respectively.
phobic chain to water (Figure S4b, see the Supporting Infor-
[
11a]
mation).
In contrast, SEM and TEM analysis showed that
amphiphilic derivative 2 formed lamellar flakes (Figure S5, see
the Supporting Information), due to the absence of the curved
structure required to form vesicular aggregates. These results
agreed with the data obtained by particle analysis.
To evaluate the potential utility of the vesicles as drug-carrier
systems, we studied their interactions with a hydrophobic dye,
Nile red, which has negligible solubility in the aqueous
medium. The emission spectrum of Nile red (c=100 mm) was
monitored for a series of solutions with varying concentrations
of conjugate 1. We observed a strong emission at l=630 nm,
which corresponded to Nile red encapsulated in the vesicles of
1 (Figure 3C). Furthermore, the emission intensity of Nile red
at l=630 nm was monitored as a function of the concentra-
tion of 1. From the inflection point of the plot, we obtained
a CAC of 0.4 mm, which is in agreement with that obtained for
1 from the absorption studies. The encapsulation of Nile in the
hydrophobic micro-environment of the vesicles of the 1 is con-
firmed by a bi-exponential emission decay profile with t=
10.05Æ0.2 and 2.1Æ0.1 ns (cf. Nile red in THF: t=4.1Æ0.4 ns).
The long-lived encapsulated Nile red species can be attributed
to storage of dye molecules inside the hydrophobic part of the
vesicles, whereas the short-lived species could be due to mol-
ecules located at the interphase between the aqueous
To understand the observation of excimer emission at
higher concentrations and the supramolecular assemblies
formed, we carried out morphological analysis of conjugates
1
and 2 through dynamic light scattering (DLS), SEM and TEM
techniques. At lower concentrations (<0.4 mm, i.e. below the
CAC), bolaamphiphile 1 showed negligible formation of nano-
aggregates (assessed by particle size analysis). Interestingly, at
the CAC (0.4 mm) we observed aggregates with a Z-average
hydrodynamic diameter of 240 nm with good correlation data
(
Figure S3, see the Supporting Information). In contrast, amphi-
philic conjugate 2 at CAC did not show good correlation data,
which indicates that the aggregates formed from this system
were not spherical. To evidence these observations, we carried
out SEM and TEM analysis and the images from both these
techniques confirmed the formation of self-assembled struc-
tures of 1 with diameters of approximately 220Æ5 nm.
To get more insight into the nature of the self-assembled
structure of conjugate 1, we carried out TEM analysis after
negative staining with phosphotungstic acid (pH 7.4). The
images obtained revealed the presence of spherical particles
with distinctive walls approximately 5 nm wide and a solid in-
terior, which confirmed that the self-assembled structures
formed were vesicular in nature (Figure 3A and b). The thick-
ness of the walls closely matched the theoretically calculated
extended-aliphatic-chain length of conjugate 1 (Figure S4a,
see the Supporting Information). The formation of vesicles can
be rationalised as a result of the cumulative effect of p-stack-
ing interactions between the NDI chromophore and the curva-
ture provided by minimising exposure of the central hydro-
[
11b]
medium and the vesicles.
Further evidence for the encapsu-
lation was obtained by observation of red-light-emitting spher-
ical particles in a solution of vesicle-encapsulated Nile red in
the aqueous medium that was examined under a fluorescence
microscope (Figure 3D).
DNA-binding properties
To investigate the potential of biomolecules as stimuli, we
studied the interactions of the conjugates with proteins [for
example, bovine serum albumin (BSA)], calf thymus (ct) DNA
and synthetic polynucleotides at different concentrations. The
successive addition of BSA (0–50 mm) led to negligible changes
in the absorption and fluorescence spectra of conjugate
1
(10 mm) in buffer (Figure S6, see the Supporting Information).
Similar observations were made for conjugate 2. These obser-
vations indicate that the conjugates undergo less-efficient in-
teractions with albumins. In contrast, the addition of ct-DNA
(
0–50 mm) in small aliquots to a solution of conjugate
(10 mm) in buffer resulted in a gradual decrease of the ab-
1
sorbance at l=382 nm, which corresponds to the NDI
chromophore (Figure 4A). Maximum hypochromicity (ꢀ42%)
was observed at a DNA concentration of 50 mm, along with
a bathochromic shift of about 3 nm with isosbestic points at
l=389 and 313 nm. The intrinsic binding constant (KDNA) was
calculated by half-reciprocal analysis (KDNA =8.61Æ0.03”
4
À1
1
0 m ), which indicates the strong binding affinity of conju-
Figure 3. A) SEM and B) TEM images of vesicles of 1 in the aqueous medium
gate 1 towards DNA.
(
c=0.4 mm). Insets: magnified images of the portion marked by the black
As the concentration of DNA increased we observed a regu-
lar and significant decrement of the fluorescence intensity at
l=390 nm (the NDI monomer) in the emission spectra of con-
jugate 1. We observed a concomitant enhancement of the
box in the respective images. C) Absorption and emission (inset) spectra of
an aqueous solution of 1 (0.4 mm) containing Nile red (100 mm). D) Fluores-
cence microscopic images of Nile red encapsulated in vesicles of conjugate
1
in the aqueous medium. lex =530 nm.
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To understand the origin of the excimer emission, we em-
ployed time-resolved emission spectroscopy (TRES). TRES ana-
lysis of conjugate 1 showed a single peak at l=395 nm (NDI
monomer) immediately after excitation (7 ps). However, in the
presence of DNA (50 mm) excimer formation was observed,
even at 63 ps, and it showed an intensity enhancement as
a function of time. After a 1 ns excitation pulse, the spectrum
was dominated exclusively by the excimer emission (lmax
=
5
10 nm). Similar observations were made for conjugate 2 in
the presence of DNA (Figure 5; Table S1, see the Supporting In-
formation).
Figure 4. Changes in the A) absorption and B) emission spectra of 1 (10 mm)
in the presence of ct-DNA in phosphate buffer (10 mm, pH 7.4) containing
NaCl (2 mm). [DNA]=i) 0 and ii) 50 mm. Insets: a) half-reciprocal analysis for
the binding of 1 with DNA and b) ratiometric plot between thee emission
maxima at l=510 and 390 nm. lex =362 nm.
emission intensity at l=510 nm, which is attributed to excimer
formation (Figure 4B). At a 50 mm DNA concentration, we ob-
served about 30-fold enhancement of the I510/I390 ratio, which
led to DNA recognition in the buffer medium through the ex-
cimer emission of conjugate 1. Similarly, we observed a signifi-
cant hypochromicity (ꢀ39%) in the absorption spectra of con-
jugate 2 (20 mm) upon addition of DNA (73 mm). In the emis-
sion spectra of 2, we observed a prominent and gradual de-
crease in the peak intensity at l=378 nm, with a concomitant
peak enhancement at l=500 nm (Figure S7a, see the Support-
ing Information). The KDNA value between conjugate 2 and
Figure 5. Time-resolved emission spectra (TRES) of conjugates A) 1 (10 mm)
and B) 2 (20 mm) in the presence of DNA (50 mm) in phosphate buffer
(10 mm, pH 7.4) monitored after an excitation pulse of i) 7 ps; 0.5 ns,
ii) 63 ps; 1.4 ns, and iii) 1.4 ns; 13.8 ns. lex =375 and 335 nm, respectively.
4
À1
DNA was 5.56Æ0.1”10 m , determined by half-reciprocal
analysis. The K value of bolaamphiphilic conjugate 1 was
[
12]
DNA
about 1.6-fold higher than that of conjugate 2. This is due to
the presence of two cationic charges in the former versus
a single cationic charge in the latter.
To evaluate the DNA-sequence selectivity, we investigated
the interactions of conjugates 1 and 2 with synthetic poly-
oligonucleotides poly(dG·dC)–poly(dG·dC) and poly(dA·dT)–
poly(dA·dT). Addition of increasing aliquots of poly(dG·dC)–
poly(dG·dC) to a solution of conjugate 1 in buffer showed an
enhancement of the excimer emission at l=510 nm (Fig-
ure S8, see the Supporting Information) and a concomitant de-
crease of the monomer emission. As in the case of ct-DNA, we
determined the association constant (KDNA =3.52Æ0.2”
To evaluate the effect of DNA on the excited-state prop-
erties, we analysed the fluorescence decay profiles of conju-
gates 1 and 2 under different conditions. The successive addi-
tion of DNA aliquots to a solution of conjugate 1 in buffer re-
sulted in a gradual increase in the fluorescence lifetime when
monitored at l=510 nm. A significant enhancement to t=
5
À1
2
.49 ns (initial <t> =0.1 ns) was observed in the presence of
10 m ). Similar observations were made for conjugate 2
5
À1
DNA (50 mm) (Figure S7b, see the Supporting Information). In
contrast, we observed negligible changes of the fluorescence
lifetimes of conjugate 2, which corresponded to monomeric
emission at l=378 nm, even at a 73 mm DNA concentration.
However, when we monitored the emission at l=500 nm we
observed a bi-exponential fluorescence decay profile with t=
(KDNA =1.71Æ0.1”10 m ). The addition of poly(dA·dT)–poly-
(dA·dT) to 1 also led to similar ratiometric changes, albeit with
4
À1
lower affinity (KDNA =1.4Æ0.2”10 m ). These results indicate
that conjugates 1 and 2 show strong sequence dependency,
and the efficiency was in the order: poly(dG·dC)–poly(dG·dC)>
ct-DNA>poly(dA·dT)–poly(dA·dT). This sequence dependency
can be attributed to the relatively low ionisation potential of
the GC pair relative to the AT sequences, as reported in the lit-
2
.5Æ0.02 (31%) and 17.2Æ0.07 ns (69%), which correspond to
the monomer and excimer of the naphthalimide chromophore,
respectively.
[
13]
erature.
17660
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[
17]
Nature of the DNA-binding interactions
binding mode, similar to reported examples.
Further, the
melting temperature of synthetic polynucleotide poly(dA·dT)–
To determine the nature of the interactions of the conjugates
with DNA, we studied the effect of the ionic strength of the
medium. With successive addition of aliquots of DNA to a solu-
tion of 1 in buffer containing NaCl (2 or 50 mm) we observed
a lower I510/I390 ratio at the higher NaCl concentration. When
the DNA-binding studies were carried out in a buffer that con-
tained NaCl (500 mm), we observed only negligible changes in
the DNA-mediated excimer emission of conjugate 1. The asso-
ciation constants for these interactions were calculated in solu-
tion in a buffer that contained NaCl (50, 100 or 500 mm) as
poly(dA·dT) (T =448C) substantially increased to 66 or 548C in
m
the presence of conjugates 1 and 2, respectively (Figure 6),
which indicated that both 1 and 2 significantly stabilise the
[
18]
duplex, predominantly through intercalative interactions.
4
À1
KDNA =1.15, 0.85 and 0.71”10 m , respectively. Similar obser-
vations were made for conjugate 2: K =0.75, 0.58 and 0.41”
DNA
4
À1
1
0 m in the presence of NaCl (50, 100 or 500 mm), respect-
ively. These observations clearly suggest that electrostatic in-
teractions play an important role in the binding interactions of
conjugates 1 and 2 with DNA.
The DNA-binding affinities of 1 and 2 were further investi-
gated by employing the competitive ethidium bromide (EB)
[
14]
binding assay.
For example, a 2.4-fold emission enhance-
ment was observed at l=630 nm after addition of ct-DNA to
EB, which corresponded to formation of an EB–DNA complex
5
À1 [14b]
(
KDNA =1.23Æ0.07”10 m ).
Subsequent addition of conju-
gate 1 to this EB–DNA complex, showed approximately a 1.7-
fold (ꢀ70%) decrease in the emission intensity (Figure S9, see
the Supporting Information). Similarly, amphiphilic conjugate 2
showed an approximately 1.5-fold (ꢀ62%) decrease in the
emission intensity of the EB–DNA complex. These results cor-
roborate that both 1 and 2 can efficiently displace EB, possibly
due to their intercalative mode of interaction.
Figure 6. A) Thermal denaturation and B) differential thermal denaturation
curves for poly(dA·dT)–poly(dA·dT) (8.3 mm), in the absence ( ; T =448C)
and presence of conjugate 1 (*; 8.3 mm, T
=668C) and 2 (~; 8.3 mm,
=548C). Absorbance monitored at l=260 nm.
&
m
m
T
m
We employed circular dichroism (CD) spectroscopy, viscosity
and thermal denaturation analysis to further understand the
nature of the interactions. The CD spectrum of ct-DNA consists
of distinctive peaks at l=280 (+ve) and 245 nm (Àve) (Fig-
ure S10a, see the Supporting Information). Upon addition of
conjugate 1 to a solution of ct-DNA in buffer, a bisignated CD
signal with maxima at l=363 (+ve) and 411 nm (Àve) was ob-
served. Both these bands are located on either side of the ab-
sorption maximum of the free conjugate 1 (l=382 nm). This
observation could be attributed to exciton coupling between
DNA-mediated disassembly of the vesicles
To evaluate the potential of DNA as a stimulus, we investigated
the interactions of DNA with vesicles of conjugate 1 formed at
the CAC (0.4 mm). Interestingly, upon addition of DNA
(0.3 mm) to vesicles of 1 in solution in buffer we observed sig-
nificant hypochromicity (ꢀ36%) in the absorption spectrum.
In the emission spectrum, we observed significant enhance-
ment of the excimer intensity (I510/I390 =1.6; Figure S11, see the
Supporting Information), which indicated disruption of the
vesicles under these conditions. To obtain evidence for this
transformation, we carried out morphological analysis of the
vesicles in the presence of DNA through DLS and TEM tech-
niques (without negative staining). As shown in Figure 7A, we
observed prominent changes in the size-distribution curves
and the amplitude of the correlogram in the presence of DNA
(0.4 mm) (Figure 7A and b; ii). Under these conditions, TEM
images indicated only reticulated fibres that were about
300 nm wide (Figure 7C), which confirmed vesicle disassembly.
Furthermore, we utilised the disruption of the vesicles to re-
lease the encapsulated dye molecules. Figure 7D shows the
changes in the absorption and emission properties of Nile red
encapsulated in vesicles of 1 in solution in aqueous medium in
the presence of DNA. After addition of DNA, the spectrum of
[
15]
the planar NDI aromatic surfaces at the intercalative sites. Al-
though the exciton CD signals are not characteristic features of
intercalators (the nearest-neighbour exclusion principle), few
classical intercalators that show distinctive bisignated bands
[15b]
are reported in the literature.
However, amphiphilic deriva-
tive 2 showed a negative ICD signal (l =344 nm), which is
max
[16]
typically observed for intercalators, with transition moments
perpendicular to the DNA longitudinal axis (Figure S10b, see
the Supporting Information).
Changes in the dynamic viscosity of DNA were monitored
with increasing conjugate concentration. We observed a value
of 1.1Æ0.02 mPas for ct-DNA (0.3 mm) in buffer at 258C,
which gradually increased to 1.57Æ0.01 and 1.43Æ0.01 mPas
in the presence of conjugate 1 (0.1 mm) or 2 (0.1 mm), respect-
ively, under identical conditions. The viscosity changes ob-
served indicate that these systems undergo an intercalative
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recorded with a Shimadzu UV-3101PC UV-Vis-NIR scanning spectro-
photometer. Fluorescence spectra were recorded with a SPEX-Fluo-
rolog F112X spectrofluorimeter. All experiments were carried out at
room temperature (25Æ18C), unless otherwise mentioned.
Materials and methods
Starting materials: NDA, NMA, 11-bromoundecanoic acid and
ethanolamine were purchased from Aldrich and S. D. Fine Chem-
icals, India.
DNA-binding studies: The DNA-binding studies were performed in
phosphate buffer (10 mm, pH 7.4) that contained NaCl (2, 50, 100
and 500 mm). KDNA was determined by a half-reciprocal plot of D/
Deap versus D [Eq. (1)] by recording the absorbance at the respect-
ive maxima after each addition of ct-DNA.
Figure 7. A) Size-distribution and B) correlation data of the conjugate
i) alone (0.4 mm) and ii) in the presence of ct-DNA (0.4 mm). C) TEM image
of vesicles of conjugate 1 in the aqueous medium under similar conditions.
D) Changes in the absorption and emission (inset) spectra of Nile red encap-
sulated in vesicles of conjugate 1 in aqueous medium upon addition of ct-
DNA [c=i) 0 and ii) 0.3 mm].
1
D=Deap ¼ D=De þ 1=ðDeKDNA
Þ
ð1Þ
D is the concentration of ct-DNA base pairs, De =[e Àe ], De=
ap
a
F
[12]
[
e Àe ], e is the apparent extinction coefficient (A
/[con-
b
F
a
max[observed]
jugate]), e is the extinction coefficient of the bound form of the
b
conjugate and e is the extinction coefficient of the free conjugate.
F
eb was determined from the gradient (1/De) and KDNA was obtained
from the ratio of the slope to the y-intercept [1/(DeKDNA)].
the vesicles that contained Nile red showed considerable
hypochromicity at l=530 nm, which corresponded to the re-
leased dye, with concomitant quenching of the fluorescence
intensity at l=630 nm. These results demonstrate the poten-
tial of vesicles of conjugate 1 as carriers of hydrophobic dye
molecules through an encapsulation–release process that uses
DNA-stimulated disassembly.
Viscometric titrations were performed by using a LAUDA DLK10
automated viscometer, thermostat controlled at 258C in a con-
stant-temperature bath. The concentration of ct-DNA was 0.3 mm,
and the flow times were measured with an automated timer. Each
sample was measured three times and an average flow time was
calculated.
Determination of the CAC: The CAC values of 1 and 2 were deter-
mined by UV/Vis spectroscopy. A stock solution of 1 or 2 in water
(
5 mm) was prepared, and from this a series of solutions of various
concentrations were made (46 mm–1.1 mm) and equilibrated for
h at RT before the analysis. The absorbance of 1 and 2 at l=380
Conclusion
2
Amphiphilic naphthalene imide conjugates 1 and 2 showed
good solubility in the aqueous medium and exhibited signifi-
and 340 nm, respectively, was plotted against concentration and
the CAC value was estimated from the inflection point.
4
À1
cant DNA association constants (KDNA =5–8”10 m ) with in-
tercalative binding interactions. Of these systems, NDI conju-
gate 1 exhibited vesicles in the aqueous medium at and above
a CAC of 0.4 mm, whereas conjugate 2 aggregated to form la-
mellar flakes. The self-assembled vesicles of 1 encapsulated
hydrophobic molecule Nile red efficiently. However, in the
presence of DNA these vesicles disassembled, which was indi-
cated by various photophysical and microscopic techniques.
Uniquely, this transformation could be effectively employed to
release the encapsulated hydrophobic molecules by using
DNA as a stimulus. Thus, these conjugates exhibited their po-
tential use as DNA probes, as well as potential carrier systems
for the delivery of hydrophobic guest molecules in the aque-
ous medium.
Encapsulation of Nile red by the vesicles of conjugate 1: A solu-
tion of Nile red in THF (3 mL, 0.1 mm) was placed in various glass
vials and the solvent was evaporated. Solutions of various concen-
trations of 1 were added to the vials that contained Nile red, and
the mixture was sonicated for 15 min and allowed to stand for 2 h
before fluorescence spectroscopic analysis (l =530 nm). The final
ex
concentration of Nile red was 100 mm and the emission intensity of
encapsulated Nile red at l=630 nm was plotted versus the con-
centration of 1 and the inflection point of the plot was taken as
the CAC of conjugate 1.
Acknowledgements
This work was supported by the Department of Science and
Technology, New Delhi and the CSIR-National Institute for
Interdisciplinary Science and Technology (CSIR-NIIST) (CSC
Experimental Section
0
134), Trivandrum. This is contribution PPG-363 from CSIR-
NIIST, Trivandrum.
General techniques
The equipment and procedures for melting-point determination
[
19]
and spectral recordings are described elsewhere.
All melting
Keywords: amphiphiles · DNA binding · naphthalene imides ·
self-assembly · vesicles
points are uncorrected and were determined with a Mel-Temp II
melting-point apparatus. The electronic absorption spectra were
Chem. Eur. J. 2015, 21, 17657 – 17663
www.chemeurj.org
17662
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Received: July 28, 2015
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969.
Published online on October 22, 2015
Chem. Eur. J. 2015, 21, 17657 – 17663
www.chemeurj.org
17663
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim