Article

pubs.acs.org/Langmuir

Spontaneous Formation of Vesicles by Sodium 2‑Dodecylnicotinate

in Water
Aparna Roy, Monali Maiti, and Sumita Roy*
Department of Chemistry and Chemical Technology, Vidyasagar University, Paschim Medinipur-721 102, India
*
S Supporting Information

ABSTRACT: The surface activity and aggregation behavior of a

synthesized nicotinic acid based anionic surfactant, sodium 2-
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dodecylnicotinate, were studied in aqueous solution. The self-

assembly formation was investigated by use of a number of
techniques, including surface tension and conductivity measure-
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ments, fluorescence spectroscopy, dynamic light scattering measure-

ment, gel permeation chromatography, and microscopy. The
amphiphile exhibits two breaks in the surface tension vs concentration plot, indicating stepwise aggregate formation and thus
producing two values of the aggregation concentration. Stepwise aggregation of the amphiphile was further confirmed by steady-
state fluorescence spectroscopy using pyrene as a probe molecule, and also the micropolarity of the aggregates was determined.
The rigidity of the microenvironment was estimated by determining steady-state fluorescence anisotropy using 1,6-diphenyl-
1,3,5-hexatriene as a fluorescence probe molecule. The average hydrodynamic radius and size distribution of the aggregate
suggest formation of larger aggregates in aqueous solution. The formation of vesicles in water was established by conductivity
measurement and a dye entrapment experiment. The entrapment of a small solute and the release capability have also been
examined to demonstrate these bilayers form enclosed vesicles. Transmission electron micrographs revealed the existence of
closed vesicles and closed tubules in aqueous solution. Therefore, for the first time, it has been observed that this simple single-
chain nicotinic acid based amphiphile spontaneously assembles to vesicles in aqueous solution.

■ INTRODUCTION
Amphiphilic molecules in aqueous solution self-assemble to
form a variety of microstructures above a certain concentration
and have been a subject of intensive research during the past
four decades.1−3 Among all the self-assemblies, vesicular
structures are most important because lipid bilayer vesicles or
liposomes have been widely studied for drug delivery and
material synthesis and as model biomembranes.4,5 Vesicles
store, transport, or digest cellular products and waste materials.
They are involved in metabolism, buoyancy control,6 and
enzyme storage. Because of their versatility, there are many
reports on vesicle formation from natural amphiphiles (mainly
phospholipids) and synthetic surfactants in the literature.7,8
There are also instances of formation of vesicular aggregates by
amphiphiles having a large headgroup size and hydrogen
bonding. Grinberg and co-workers9 have reported that a triple-
headed derivative of vernonia oil assembles to vesicles in the
presence of cholesterol. Cantu et al.10 have shown that the
vesicles made from ganglioside GM3 are stabilized in the
presence of the larger headgroup ganglioside amphiphile GM1.
A new class of nucleoamphiphiles assemble in water to form a
nano- to micrometer left-handed helix with stacked bilayer-type
aggregates induced by simply complexing chiral GMP or AMP
(guanosine 5′-monophosphate or adenosine 5′-monophos-
phate) with a nonchiral monocationic amphiphile having two
hydrophobic chains of 12 and 14 carbons (dialkyldimethy-
lammonium bromide).11 In spite of all the reports, there is not
a single paper on the formation of a vesicular structure by a
surfactant derived from nicotinic acid having a simple aliphatic
chain as a hydrophobic moiety. We became interested in the
nicotinic acid derived surfactant as it is well-known to all that
pyridine is an important model molecule in biology as many
drugs and metabolites are pyridine based.12 Pyridine and
pyridinium salt both are used as ligands, and pyridine itself is a
useful solvent in synthetic chemistry.13 “Niacin”, which is
nothing but simple nicotinic acid, is widely known as vitamin
B3. N-Methylnicotinic acid (trigoneline) plays an important
biological role in controlling the G2 factor mechanism.14,15
Kalyanasundaram et al. have reported the critical micellar
concentration and critical micellar temperature for various 1-
alkyl-3-carbamoylpyridinium halide surfactants (N-alkylnicoti-
namides) in aqueous solution.16 The authors also studied the
micellazation properties of N-alkylnicotinic acid surfactants in
aqueous solution.17 Chevalier et al. have investigated the
micellar properties and mesophase structure of two isomers of
N-alkylpyridiniocarboxylates with their carboxylate group at
position 3 or 4 on the pyridinium ring.18 Jiang and co-workers
have synthesized and studied the surface-active properties of a
novel class of gemini pyridinium surfactants having a 4-
methylene spacer group, and also they investigated the
interactions of these amphiphiles with polyacrylamide
(PAM).19 Therefore, in this work, we have attempted to
Received: December 13, 2011
Revised: August 1, 2012
Published: August 8, 2012

© 2012 American Chemical Society 12696 dx.doi.org/10.1021/la302484x | Langmuir 2012, 28, 12696−12703
Langmuir
synthesize and characterize the vesicles formed by a simple
single-chain anionic surfactant derived from nicotinic acid in
aqueous solution.
■
RESULTS AND DISCUSSION
We have performed the synthesis of a new nicotinic acid based
surfactant named sodium 2-dodecylnicotinate, abbreviated as
SDDNA (Figure 1) according to the scheme given in the
Supporting Information. The molecular structure of the
compound was identified by FT-IR, 1H NMR, and LC−MS
(see the Supporting Information).
Figure 1. Chemical structure of SDDNA.
Surface Tension Study. It is well-known that, among all
the available methods, the surface tension method is the most
useful and accurate method for determining the critical
aggregation concentration. Therefore, this method was used
to determine the critical aggregation concentration (CAC) of
SDDNA. A plot of surface tension (γ) versus log-
(concentration, C) in aqueous solution (pH 6.70) of the
amphiphile is shown in Figure 2.
Figure 2. γ vs log C of SDDNA in aqueous solution.
It is clear from the figure that there are two break points of
the plot γ vs log C corresponding to two CAC values. As the
number of points corresponding to the first break point is
fewer, we thought this might be due to the presence of some
surface-active impurity in the compound. However, no
minimum was observed around the second break point of the
surface tension plot, which ruled out the presence of any
surface-active impurity in the amphiphile.20 Furthermore, the
purity of the sample was confirmed by 1H NMR and LC−MS.
This type of two break points due to post micellar aggregation
has also been reported for other surfactants,20−27 mixed
surfactants,28 and phenyl ring bearing cationic surfactants.29
The CAC values and all the interfacial properties are given in
Table 1.
The CAC and surface tension at the CAC (γCAC) values are
lower than those of the corresponding fatty acid soaps, which
suggests that the amphiphile is a better surfactant than
conventional soaps.30,31 The good surface activity is also
reflected by a larger pC20 (>3)32(where pC20 = negative
logarithm of the surfactant concentration required to reduce
the surface tension of water by 20 units). On the other hand,
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Table 1. Surface Properties of SDDNA
property value property value
CAC (mM) 0.030, 0.0240,a 0.320, a0 (nm2 1.070
0.340a molecule−1)
γCAC (mN m−1) 43, 36.600 ΔGa° (kJ mol−1) −27.540
ΠCAC (mN m−1) 33.900 ΔGad° (kJ mol−1) −49.550
α 0.641 CAC/C20 35.660
Γ2 × 106 (mol 1.540 pC20 5.0450
m−2)
a
Data are obtained from fluorescence measurements.
the ratio CAC/C20, which is a measure of the tendency of the
surfactant to adsorb at the air−water interface relative to the
formation of the aggregates, is greater than those of
conventional hydrocarbon monomeric surfactants. The max-
imum surface excess concentration (Γ2) and minimum surface
area per surfactant molecule at the air−water interface (a0)
were also estimated from the surface tension plot using the
Gibbs adsorption equation:33
Γ2 = −(1/2.303nRT )(dγ /(d log C)) (1)
18
a0 = 10 /(Γ2NA ) (2)
where dγ/(d log C) is the maximum slope, NA is Avogadro’s
number, T is the absolute temperature, and n is 2 for dilute
solutions of monovalent ionic surfactants.34
For SDDNA, the a0 value thus obtained is in the range 1.0
nm2 ≤ a0 ≤ 1.1 nm2, which is indicative of the formation of
bilayer aggregates.25 The standard free energy of aggregation
per mole of surfactant was determined by using the equation22
ΔGa° = RT(1 + β) ln CAC (3)
where β = 1 − α, in which α is the degree of micellar ionization
which can be determined from the ratio of the slope of the
conductivity versus concentration lines above and below the
CAC (for the plot see the Supporting Information).
ΔGad° was evaluated from the equation35
ΔGad° = ΔGa° − πCAC/Γ2 (4)
where πCAC is the surface pressure (= γ0 − γCAC, γ0 being the
surface tension of pure water). The ΔGa° and ΔGad° values are
also listed in Table 1. The large negative value of ΔGa° suggests
spontaneity of the aggregate formation in water.
Microenvironment Study. To investigate the micro-
environment of the self-assembly, fluorescence studies were
performed using pyrene and 1,6-diphenyl-1,3,5-hexatriene
(DPH) as an extrinsic fluorescence probe because both the
probe molecules bind preferentially to the hydrophobic region
of the self-assemblies. We know that the ratio of the intensities
corresponding to the first and third vibronic bands (I1/I3) is
sensitive to solvent polarity.36 Its value is maximum in water
and decreases with a decrease in solvent polarity. Therefore, it
has been widely used as a micropolarity probe for self-
assemblies. The I1/I3 ratio was measured in the presence of
various surfactant concentrations. A two-step change of the plot
I1/I3 vs log(concentration) of the surfactant was obtained
(Figure 3), which is consistent with the surface tension plot.
Two CACs and the polarity ratio corresponding to two
CACs thus obtained are summarized in Table 2. The I1/I3 ratio
above the first CAC is 1.77 (inset of Figure 3), which is very
close to that of water (1.86), indicating that the probe molecule
is partially exposed to the bulk solvent, which is only possible if
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the formation of bilayer aggregates.21,40,41 The high anisotropy

value (r = 0.108) and high micropolarity index (I1/I3 = 1.77)
just above the first CAC indicates formation of a flat bilayer
structure above the first break point. The anisotropy value
gradually increases and remains constant up to 1.25 mM
(greater than the second CAC). On the other hand, the
micropolarity index gradually decreases to a very low value (I1/
I3 = 1.23). These two phenomena suggest that the flat bilayer
aggregates transformed into closed vesicles and tubules. After
an amphiphile concentration of 1.5 mM the fluorescence
anisotropy value of SDDNA decreases instantaneously,
indicating closed structures are malformed to open tubules
Figure 3. Polarity ratio I1/I3 vs log C. Inset: I1/I3 vs log C for the first and/or cylindrical micelles.
break point. Dynamic Light Scattering. The dynamic light scattering
method was employed to determine the hydrodynamic radius
Table 2. Self-Assembly Properties of SDDNA of the aggregates formed by SDDNA in aqueous solution
because it is an excellent technique for distinguishing bilayer
property value property value
structures such as vesicles, lamellae, and tubules from micelles
I1/I3 1.77, 1.23 Nagg × 10−4 22.92 on the basis of the size of the aggregates. The z-average
Rh (nm) 98.84 D × 1012 (m2 s−1) 2.24 hydrodynamic radius (Rh) of a 1 mM aqueous solution was
found to be 98.84 nm, which suggests the existence of larger
the probe molecule is solubilized in a nonspherical aggregate aggregates in solution (see the Supporting Information for the
such as a flat bilayer as indicated by the a0 value. The polarity size distribution). The apparent diffusion coefficient (Dapp) of
ratio above the second CAC is very low, which suggests that the the amphiphile was calculated using the Stokes−Einstein
probe molecule is solubilized in spherical aggregates. A similar equation:
type of result has also been reported by Dey et al.21,40 This is
due to the fact that pyrene molecules are nonpolar in nature, Dapp = kBT /6πηR h (5)
are generally soluble in the hydrocarbon layer, and encounter
only a few or no water molecules around them in the case of Rh and Dapp values thus obtained are listed in Table 2. The Dapp
spherical aggregates. Also the lower micropolarity value of the value is much smaller than that of normal spherical micelles
secondary aggregates compared to that of primary aggregates (∼10−10 m2 s−1).26 The mean aggregation number (Nagg) of the
indicates more ordering at the interface, which may be due to aggregates (assuming a bilayer vesicle) was also estimated by
the formation of a vesicular structure above the second CAC. use of the equation42
To further shed light on the structural change at
concentrations above the two break points, we measured the Nagg = 8πR h 2/a0 (6)
steady-state fluorescence anisotropy (r) using DPH as a
The large Rh and Nagg values with a very small (1/100 times)
fluorescence probe because it is a well-known membrane
diffusion coefficient compared to that of a normal spherical
fluidity probe and different scientists have used it to study many
micelle suggest that the aggregates formed by SDDNA are very
lipid bilayer membranes.37−39 r is an index of the equivalent
large.
microviscosity (microfluidity) of the vesicle core. Therefore, r
Conductivity Study. To demonstrate the transformation of
was measured at different concentrations above and below the
flat bilayer aggregates to a closed vesicle, the conductivity of a 1
second CAC. It has been observed that the anisotropy value
mM KCl solution was measured in the presence of a 1 mM
increases from 0.06 mM (above the first break point) and
SDDNA solution. It was observed that the sum of the
remains almost unchanged up to a concentration of 1.5 mM (r
conductivity values of a 1 mM KCl solution (160 μS cm−1) and
= 0.159). After this, the anisotropy value decreases and
a 1 mM SDDNA solution (77 μS cm−1) is 37 μS cm−1 greater
becomes almost constant at higher concentration. The r value
than that of 1 mM KCl containing a 1 mM (greater than the
for different concentrations of SDDNA is given in Table 3.
second CAC) SDDNA solution (200 μS cm−1). On the other
We are unable to measure the anisotropy below the first
hand, there was no decrease of conductivity of the 1 mM KCl
CAC because the intensity of DPH in the solutions was very
solution in the presence of 0.15 mM (less than the second
low. The value of anisotropy is greater than that of the lecithin
CAC) SDDNA. This study clearly indicates the existence of
liposomes (r ≈ 0.098) but less than that of sphingomyelin
vesicles after the second break point. A similar type of decrease
liposomes (r ≈ 0.247).37 The large value of r is due to tight
of the conductivity value of the KCl solution due to formation
packing of the hydrocarbon chains and perhaps is indicative of
of vesicles has been reported by Hoffmann43 and Dey et al.21
Actually the vesicles are able to entrap a part of the solvent and
Table 3. Anisotropy Values of SDDNA at Different the salt. As a result they prevent the entrapped charge carriers
Concentrations (K+ and Cl− ions) from contributing to the conductivity of the
solution, and thus, the conductivity of the vesicular solution
concn (mM) r concn (mM) r concn (mM) r
becomes lower than that of the conductivity of the salt solution.
0.06 0.108 1 0.163 2.25 0.123 The variation of the conductivity change (Δκ) with surfactant
0.08 0.137 1.25 0.162 2.50 0.117 concentration is shown in Figure 4.
0.20 0.156 1.50 0.159 2.75 0.108 The value of Δκ at first increases with the surfactant
0.40 0.160 1.75 0.144 3 0.107 concentration and then decreases. This is because of the fact
0.70 0.162 2 0.132 that the population of vesicles increases with the surfactant
12698 dx.doi.org/10.1021/la302484x | Langmuir 2012, 28, 12696−12703
Langmuir
Figure 4. Δκ vs surfactant concentration.
concentration and more and more K+ and Cl− ions are being
entrapped inside the aqueous core. As a result the Δκ value
increases. The decrease in the Δκ value at higher surfactant
concentrations is most probably due to the transformation of
closed vesicles to open tubules and/or cylindrical micelles,
which causes release of the entrapped water with salt.
Entrapment of Methylene Blue in Vesicles. Further-
more, to confirm the formation of vesicles from this newly
synthesized amphiphile, we employed dye entrapment studies
to check whether the aggregates contain closed inner aqueous
compartments, because micelle-forming amphiphiles in water
have no capacity to entrap hydrophilic water-soluble molecules
(dye). Walde and Namani44 have used arsenazo III as a water-
soluble dye to establish the aqueous core of decanoic acid/
dodecylbezenesulfonate vesicles. A similar type of experiment
was also performed by Dey et al.40 to confirm the inner
aqueous compartments of vesicles formed by N-[4-(n-
dodecyloxy)benzoyl]-L-aminoacidates in aqueous solution
using methyl orange as a dye molecule. Gokel et al.45 showed
that the aggregates of steroidal azacrown derivatives in aqueous
media possessed entrapment capacities. Riboflavin was used as
a dye molecule by Bhattacharya and co-workers to establish the
vesicular structure formed by cationic mixed-chain surfactants46
in 1995 and hybrid bolaphile/amphiphile ion pairs47 in 1998.
They have also used methylene blue (MB) as a water-soluble
dye to establish inner aqueous compartments of some
synthesized click adducts48 and sugar-based amphiphiles.49
Therefore, to investigate the entrapment capacity of the
aggregate generated from the amphiphile SDDNA, we chose
MB as a dye molecule. To achieve our goal, we prepared a 1
mM stock solution of SDDNA in water containing 0.1 mM
MB. The resulting solution (2 mL) was then loaded into a gel
filtration column (preequilabrated Sepharose 4B) and eluted
with triply distilled water. Translucent vesicular suspensions
eluted right after the void volume. The gel filtration was
continued until free MB was gel filtered. The separate fractions
of each were collected (2 mL each), and the amount of dye
present was evaluated by the spectrophotometric method. The
profile of gel filtration was obtained by plotting the absorbance
at 665 nm of all the fractions obtained from the gel filtration
column vs the elution volume (Figure 5). It was observed that
there was a small initial portion containing vesicles entrapping
approximately 1.79% of the total dye followed by a large peak
which was due to the free, unentrapped dye.
Kinetics of Transmembrane Permeation. To shed light
on whether the bilayer membranes of the vesicles are
permeable, a kinetic study was performed fluorometrically
because of all the methods available to study the membrane
permeability, methods utilizing the fluorescence probe are
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Figure 5. Gel filtration profile of the separation of the dye entrapped
(small peaks) in a vesicle of SDDNA from the corresponding free dye
(methylene blue).
widely used due to their simplicity and convenience.50 A
number of probes such as pyranine,51 calcein,52 carboxyfluor-
escein,53 riboflavin,46,47 etc. have been used to estimate
membrane permeability. Here we used riboflavin as a probe
molecule because it is a neutral water-soluble molecule which is
strongly fluorescent in the neutral form and becomes
nonfluorescent upon deprotonation (pKa of riboflavin ∼10.2).
It can also withstand a low ionic strength. This probe has also
been successfully used by different scientists54−56 for the
determination of transmembrane pH gradients with unrelated
vesicle-forming surfactants of diverse surface charges. The
fluorescence intensity due to the membrane-bound and
entrapped riboflavin was measured at 514 nm (λex = 374
nm). This includes the dye molecules that were adsorbed on
the outer membrane surfaces and the dye molecules that were
entrapped within the inner water pools. When the pH of the
vesicle dispersion was increased from 6.8 to 10.2, the
fluorescence intensity of the riboflavin decreased initially
“instantaneously” to about 60 ± 5% of the original value at
pH 6.8. This characteristic of immediate capacity (40 ± 5%)
loss in the fluorescence intensity is due to the deprotonation of
riboflavin molecules that are bound at the outer surfaces of the
vesicles. This result (decrease of the fluorescence intensity of
riboflavin due to the conversion of its neutral form to the
deprotonated form upon pH adjustment from 6.8 to 10.2)
established that it contained an internal aqueous compartment.
The residual fluorescence intensity disappeared as a function of
time (Figure 6) since instantaneous ionization of the riboflavin
molecules occurred at the exposed exovesiculer surfaces as the
pH was increased.
Since the fluorescence intensity disappeared as a function of
time, we calculated the half-time of the process (t1/2 ≈ 1.52
Figure 6. OH− permeability profile of riboflavin-entrapped vesicular
SDDNA.
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Figure 7. Negatively stained (with 2% aqueous uranyl acetate solution) TEM micrographs of (A−C) 0.7 mM (inset of (C), vesicle with wall
thickness of 3.18 nm; the bar represents 50 nm), (D) 1 mM, (E) 1.25 mM, (F, G) 1.50 mM (inset of (G), tubule with closed end), and (H, I) 2 mM
(inset of (H), open tubules) SDDNA in water.

min, kperm ≈ 7.58 × 10−3 s−1). Taking the bilayer thickness of

the amphiphile as 3.18 nm (obtained from a TEM micrograph,
Figure 7C) and assuming the time-dependent loss of
fluorescence intensity by exovesicular OH− as a permeation-
limited deprotonation of riboflavin entrapped in the internal
aqueous compartment, we calculated P ≈ 1.53 × 10−9 cm/s for
the permeation constant of OH− toward vesicles of SDDNA at
pH 10.2.
TEM. The transmission electron microscopy technique was
used to visualize the actual morphology of the aggregates in
dilute and concentrated solutions of the amphiphile. TEM
pictures of negatively stained specimens prepared from different
aqueous SDDNA solutions are shown in Figure 7. The
transmission electron micrographs of a 0.7 mM (just above 2
times the second CAC) aqueous solution of SDDNA (Figure
7A−C) clearly exhibit formation of closed vesicles in an
aqueous solution of the amphiphile having an internal diameter
in the range of 30−100 nm. The inner and outer diameters of
the vesicle are 79.92 and 86.28 nm, respectively. Therefore, the
wall thickness of the lamella is about 3.18 nm (inset of Figure
Figure 8. Schematic representation of bilayer formation of SDDNA.
7C).
As the hydrocarbon chain of the amphiphile has a length of At a concentration of 1 mM, the size of the vesicles increases
1.74 nm (obtained from the energy-minimized structure via the to ∼200 nm. The size of the vesicles is enhanced to ∼325 nm at
Hartree−Fock method by Gauss View 3.0), each lamella should a 1.25 mM amphiphile concentration. At a concentration of
have a thickness of about 3.48 nm. However, the length of the 1.50 mM, not only does the size of the vesicle become very
wall thickness is smaller than twice the length of the large (∼750 nm, Figure 7F) but also the vesicles connect to
hydrocarbon chain but much larger than the length of a single each other to form elongated aggregates and closed tubules
hydrophobic tail. This implies that the aggregate has ((Figure 7G). However, the micrographs (Figure 7H,I)
interdigitated hydrocarbon tails in the bilayer vesicles (Figure corresponding to 2.0 mM SDDNA solutions show the existence
8). of open tubules20,40 and cylindrical micelles.57 Therefore, the
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stable range of the vesicles is 0.7−1.25 mM. These results are in
good agreement with the conductivity and steady-state
fluorescence anisotropy measurements.
■
CONCLUSION
In summary, we have synthesized a single-chain nicotinic acid
based surfactant which is a useful surface-active agent and
spontaneously forms vesicular structures in water. To our
knowledge, this is the first report of formation of vesicles by a
nicotinic acid based amphiphile. Surface tension measurements
at different surfactant concentrations indicate stepwise
aggregate formation and thus produce two values of
aggregation concentration. Stepwise aggregation of the
amphiphile has been further confirmed by the steady-state
fluorescence spectroscopy method. The microenvironments of
the bilayer self-assemblies are nonpolar in nature and are highly
ordered. DLS measurement suggests the existence of larger
aggregates in aqueous solution. Gel permeation chromatog-
raphy and conductivity study indicate formation of closed
vesicles in solution. Kinetic study by the fluorometric method
suggests the bilayer membrane of the vesicles is permeable.
TEM pictures revealed the existence of closed bilayer vesicles
and tubules in aqueous solution. Thus, the amphiphile may
have potential use in surfactant chemistry and the pharma-
ceutical industry as a drug delivery vehicle. The amphiphile can
also be used as a useful ligand to form complexes.
■
EXPERIMENTAL SECTION
General Procedures. 2,5-Dibromopyridine, pyrene, DPH, uranyl
acetate, and Sepharose 4B were purchased from Aldrich Chemicals,
and 1-bromododecane, riboflavin, and methylene blue were purchased
from SRL. Pyrene, DPH, and riboflavin were used after recrystalliza-
tion from ethanol. Other chemicals were procured locally and used as
they were obtained. All organic solvents were purchased from Merck
and were distilled and dried as required. Aqueous solutions were made
by triply distilled water. 1H NMR spectra were recorded using a
Bruker 400 MHz instrument. LC−MS spectra were taken in a Waters
2996 machine. UV−vis absorption spectra were obtained with a
Shimadzu (model 1601) spectrophotometer. The pH measurement
was done with a pH meter, model pH LI613 (ELICO), using a glass
electrode. All measurements were carried out at room temperature
(∼30 °C) unless otherwise mentioned.
Preparation of Aggregates. A weighed amount of amphiphile
was dissolved in a certain amount of triply distilled water and shaken
by hand to form a homogeneous mixture. The mixture was left for 4 h
and afforded approximately spherical and tubular aggregates as
evidenced from transmission electron microscopy.
Surface Tension Measurement. Surface tension measurement
was carried out with a surface tensiometer (manual) using the Du
Nuoy ring detachment method. Before each experiment, the
platinum−iridium ring was thoroughly cleaned with ethanol−HCl
solution and burned in an oxidizing flame by use of a Bunsen burner.
The instrument was calibrated, and the accuracy was checked by
measuring the surface tension of triply distilled water. A 1 × 10−3 M
stock solution of the surfactant was prepared. The surface tension (γ)
was measured at different concentrations by adding a subsequent
volume of stock solution to a beaker containing a known volume of
water until the value almost reached saturation. For each
concentration, the solution was equilibrated for 15 min, and the
average of three readings was taken. The CAC was then obtained from
the break point of the plot of γ versus log C.
Fluorescence Measurements. The steady-state fluorescence
spectra of the pyrene probe (∼2 × 10−7 M) were measured on a
Hitachi F-7000 spectrophotometer optical system equipped with a 150
W Xe lamp. For the pyrene probe, a saturated solution of pyrene was
prepared in water. A weighed amount of surfactant was then added to
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the saturated aqueous probe solution. The samples were excited at 335
nm, and the emission spectrum was recorded between 350 and 550
nm with excitation and emission slit widths both set at 1 nm. Each
spectrum was blank subtracted and was corrected for lamp intensity
variation during measurement. The steady-state fluorescence aniso-
tropy (r) of DPH was obtained in the same instrument equipped with
filter polarizers that use the L format configuration. DPH was excited
at 350 nm and the fluorescence intensity was measured at 450 nm with
band passes of 2.5 and 5 nm, respectively. The fluorescence anisotropy
value was calculated by the equation
r = (I − GI⊥)/(I − 2GI⊥) (1)
where I∥ and I⊥ are the fluorescence intensities polarized parallel
(excitation and emission polarizers at 0°) and perpendicular
(excitation polarizer at 0° and emission polarizer at 90°) to the
excitation light. G is the instrumentation correction factor (G = i90−0/
i90−90).
For anisotropy measurements a 2 mM solution of the probe was
prepared in a 20% methanol−water mixture. The final concentration
of the probe was adjusted to 2 μM by addition of the appropriate
amount of stock solution.
Conductivity Measurements. Conductivity measurements were
performed with a digital conductivity meter (Systronics, model 304)
with a cell constant of 1.05 cm−1. A 1 × 10−3 M stock solution of the
surfactant was prepared. The conductivity was measured at different
concentrations by adding a subsequent volume of stock solution to a
beaker containing a known volume of water. For each measurement,
the solution was equilibrated for 5 min to obtain a constant
conductivity value. For the entrapment experiment, a 1 mM KCl
solution was prepared in triply distilled water. For each concentration,
a weighed amount of the amphiphile was dissolved in triply distilled
water and the exact same amount of the amphiphile was dissolved in
the prepared 1 mM KCl solution. All the solutions (KCl solution,
amphiphile in water, and amphiphile in KCl solution) were left for 4 h
for equilibration before measurement. After equilibration, the
conductivity of all the solutions was measured in a digital conductivity
meter.
Dye Entrapment Studies. For the dye entrapment study, a 1 mM
stock solution of the amphiphile was prepared in triply distilled water
containing 0.1 mM methylene blue (λmax = 665 nm), which is a water-
soluble dye, and the solution was left for 4 h for spontaneous vesicle
formation. A 2 mL volume of the resulting solution was then loaded
into a column packed with a pre-equilibrated Sepharose 4B matrix (25
cm height and 1.2 cm diameter) and eluted with triply distilled water.
The fractions containing vesicles were pulled out. The eluent was
collected in 2 mL fractions. The absorbance for all the fractions
containing solutions was determined at 665 nm and plotted against the
elution volume.
Kinetics of Transmembrane Permeation. An aliquot of 1 mL
(pH 6.8) was taken in a fluorescence cuvette, and the time-dependent
decrease of the fluorescence intensity at 514 nm (λex = 374 nm,
excitation and emission slit widths of 5 and 10 nm, respectively) at
∼30 °C was followed using a Hitachi F-7000 spectrophotometer upon
adjustment of the pH from 6.8 to 10.2 by the addition of an aliquot of
48 μL of a 0.035 M KOH solution. Since riboflavin fluorescence
decreases upon ionization in an alkaline medium (pKa ≈ 10.2), the
time-dependent quenching of the fluorescence intensity at 514 nm due
to riboflavin under an imposed transmembrane pH gradient of 3.4 pH
units was taken as a measure of OH− permeation across the bilayer
vesicles. The rate constant was obtained from the monoexponential
time-dependent portion of the loss of the fluorescence intensity. The
rate constant value represents an average of three independent
experiments.
DLS Studies. The size of the aggregates was determined from DLS
measurement by using a Zetasizer Nano S (ZEN 1600) light scattering
apparatus (Malvern Instruments, Westborough, MA) with a He−Ne
laser (632.8 nm, 4 mW). The Nano S (ZEN 1600) instrument
incorporates noninvasive backscatter (NIBS) optics with a detection
angle of 173°. For DLS measurements, an aqueous solution of the
dx.doi.org/10.1021/la302484x | Langmuir 2012, 28, 12696−12703
Langmuir
amphiphile was prepared in triply distilled water. The solution was
filtered directly into the scattering cell through a Whatman syringe
filter (sterile and endotoxin free, 0.2 μm). Before measurement, the
scattering cell was rinsed with the filtered solution and the sample
solution was equilibrated for 5−10 min in the DLS optical system. The
data acquisition was carried out for 10 min.
Transmission Electron Microscopy Studies. Transmission
electron microscopic measurements were performed with a high-
resolution transmission electron microscope (JEOL-JEM 2100, Japan)
operating at 200 keV. The transmission electron microscopic
measurements were performed using the aqueous solutions of the
amphiphile after equilibration for 4 h. For all the cases the surfactant
solutions were filtered by use of a Whatman syringe filter (sterile and
endotoxin free, 0.2 μm). A drop of the surfactant solution was put on a
copper grid coated with carbon and allowed to soak for 1−2 min; the
surface solvent on the grid was removed by tapping in a filter paper
followed by staining with 2% aqueous uranyl acetate solution. The
specimens were then dried in desiccators until measurement.
■
ASSOCIATED CONTENT
* Supporting Information
S Savarino, P.; Barni, E.; Pellizzetti, E.; Graetzel, M. Micellar properties
Synthesis scheme, all spectral data, conductivity as a function of
the surfactant concentration, and cumulant distribution of the
DLS study. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: rsumita4@yahoo.co.in.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by a grant from the Department of
Science and Technology (SR/FT/CS-026/2008). A.R. thanks
the University Grants Commission (Grant F.17-130/98(SA-I))
and M.M. thanks the Council of Scientific and Industrial
Research (Grant 09/599(0044)/2011-EMR-I) for research
fellowships. We thank Dr. Sajal Kanti Mal for his valuable
suggestion for synthesis of the amphiphile and Dr. S. Patra,
Government College of Engineering and Ceramic Technology,
Kolkata, for his assistance with the DLS measurement.
■
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