Spontaneous formation of pH-sensitive, stable vesicles in aqueous solution of N-[4-n-octyloxybenzoyl]-l-histidine

Article abstract of DOI:10.1039/c000898b

A new amino acid-derived surfactant, sodium N-(4-n-octyloxybenzoyl)-l- histidinate (SOBH), was synthesized. The self-assembly properties and microstructure formation of SOBH was investigated by use of a number of techniques, such as surface tension, fluorescence spectroscopy, conductivity, dynamic light scattering (DLS), confocal fluorescence microscopy (CFM), and transmission electron microscopy (TEM). The amphiphile exists in both anionic and zwitterionic forms depending upon the pH of the solution and has very low critical aggregation concentration (CAC). The results of fluorescence probe studies and TEM as well as CFM pictures showed formation of vesicles in solution of pH > 7.5. The mean size and size distribution of the vesicles was measured by the DLS technique. The interaction of the amphiphile with cholesterol was also examined. In the presence of cholesterol the vesicle size and stability was observed to increase. Effects of salt, surfactant concentration, and temperature on the vesicle formation were investigated with or without varying amounts of cholesterol. Fluorescence anisotropy data indicated that the packing in bilayers becomes tighter by inclusion of cholesterol, which increased the stability of vesicles. The vesicles formed by SOBH were also found to be stable at body temperature.

Full text of DOI:10.1039/c000898b

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PAPER
www.rsc.org/softmatter | Soft Matter
Spontaneous formation of pH-sensitive, stable vesicles in aqueous solution of
N-[4-n-octyloxybenzoyl]-L-histidine†
*
Trilochan Patra, Sampad Ghosh and Joykrishna Dey
Received 14th January 2010, Accepted 19th April 2010
DOI: 10.1039/c000898b
A new amino acid-derived surfactant, sodium N-(4-n-octyloxybenzoyl)-L-histidinate (SOBH), was
synthesized. The self-assembly properties and microstructure formation of SOBH was investigated by
use of a number of techniques, such as surface tension, fluorescence spectroscopy, conductivity,
dynamic light scattering (DLS), confocal fluorescence microscopy (CFM), and transmission electron
microscopy (TEM). The amphiphile exists in both anionic and zwitterionic forms depending upon the
pH of the solution and has very low critical aggregation concentration (CAC). The results of
fluorescence probe studies and TEM as well as CFM pictures showed formation of vesicles in solution
of pH > 7.5. The mean size and size distribution of the vesicles was measured by the DLS technique.
The interaction of the amphiphile with cholesterol was also examined. In the presence of cholesterol the
vesicle size and stability was observed to increase. Effects of salt, surfactant concentration, and
temperature on the vesicle formation were investigated with or without varying amounts of cholesterol.
Fluorescence anisotropy data indicated that the packing in bilayers becomes tighter by inclusion of
cholesterol, which increased the stability of vesicles. The vesicles formed by SOBH were also found to
be stable at body temperature.
cholesteryl hemisuccinate,18 sodium cholate,19 fatty acid with
phosphatidylethanolamine,20 diacylsuccinylglycerols20
and
Introduction
Colloidal self-assemblies composed of N-acyl-amino acid
surfactants (NAAS) are a suitable model system for investigating
several phenomena of interest to chemists and biologists because
they are mild, non-irritating to human skin and highly bio-
friendly. For these reasons they are commonly used as detergents
and foaming agents, as well as in shampoos, cosmetics, paints,
coating materials, drug formulation, drug and gene delivery and
for oil extraction. For example, micelles,1,2 vesicles,3,4 or fibers,5
that are sensitive to changes in pH, salt, temperature, or UV light
could be potentially used as templates for materials synthesis,6 or
drug carriers and delivery devices.7 Of particular interest are pH-
sensitive vesicles, whose closed bilayer structure as well as
hydrophilic core allows for the encapsulation and targeted
release of drug and DNA molecules.8 Hence, depending on the
application, efforts have been directed toward the formulation of
vesicles that are responsive to changes in pH. There are a few
reports in the recent literature that describe pH-responsive
vesicle formation by some amphiphilic molecules. Usually
a bilayer of the pH-responsive vesicles/liposomes contains pH-
sensitive amphiphiles having ionisable acidic, amine, imino or
hydroxyl groups. For example, the pH-responsive character of
the vesicles formed by amino acid-based amphiphiles,9–11 phos-
phate group-containing amphiphiles,12 sugar-based gemini
surfactants,13 fatty-acid-based amphiphiles,14 alkyl amine
oxides,15 N-stearoylcysteamine,16 N-palmitoyl homocysteine,17
tocopherol hemisuccinate21 have been reported. In these systems
and in some cases like ion-pair amphiphiles, the pH sensitivity
depends on charge as well as the chain length of the amphi-
philes22 which have the ability to destabilize the lipid bilayer
when exposed to changes in the pH environment. The pH-
induced vesicle-to-micelle transition has potential applications in
drug delivery.19,23–25 Di Marzio et al. showed the surfactant
vesicle pH-sensitivity and cytoplasmatic delivery of drugs to
target cells.18,26 Some amphiphilic pH-sensitive vesicles are used
for the controlled release of trapped materials from the inner
aqueous core of the vesicle.27,28 Similarly, vesicles which respond
to the endosomal pH range are mostly used for efficient gene
transfection.29–31 Recently Dowling et al. created a vesicle-loaded
gel to show the pH sensitivity.32
Derivatives of histidine are unusual surfactants in that the
hydrophilic portion of the molecule is relatively large and
contains both an anionic group (COOꢀ) and a cationic group
(imidazole side chain), which gives this molecule an amphoteric
nature by protonation-deprotonation to exhibit physicochemical
behavior which is not typical of that exhibited by most NAAS.33
Derivatives of histidine that respond to local pH changes in the
body are helpful for solid tumor treatment.2 Cationic amphi-
philes containing histidine functionalities in their head group are
serum compatible, nontoxic and endosome-disrupting, thus they
can be used as a synthetic nonviral gene carrier.34 In some cases
amphiphiles of histidine also form translucent gels, fibrous
structures or helical rods either in water or in water–ethanol
mixtures.35,36 The stereoselective deacylation of long-chain
amino acid esters by chiral co-micelles of N-acyl-L-histidine and
various cationic surfactants has been reported.37 Antioxidant
activity toward lipid peroxidation and the excellent emulsifying
Department of Chemistry, Indian Institute of Technology, Kharagpur, 721
302, India. E-mail: joydey@chem.iitkgp.ernet.in; Fax: +91-3222-255303;
Tel: +91-3222-283308
† Electronic supplementary information (ESI) available: Optical
microscope images and size distribution profiles of the aggregates. See
DOI: 10.1039/c000898b
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activity of N-acyl-L-histidine have also been reported.38 This
attracted our attention toward the self-assembly properties of
histidine-containing amphiphiles. Recently, we have shown that
4-(N-n-alkyloxybenzoyl)-L-histidine amphiphiles produce ther-
moreversible, pH-responsive hydrogels in buffered water at
different pH.39
sodium salt of the compound (SOBH) was obtained by reacting
N-[4-n-octyloxybenzoyl]-L-histidine with sodium methoxide in
methanol and finally recrystallized from the acetone–water
mixture. Chemical identification of the compound was per-
formed by use of 1H NMR and FT-IR spectroscopy.
In this work, we have investigated the aggregation behavior of
sodium 4-(N-octyloxybenzoyl)-L-histidinate (SOBH) in dilute
aqueous solutions. Earlier work in our laboratory has shown
that this group of amphiphiles, with an amino acid head group,
spontaneously forms vesicles and chiral helical aggregates in
water.3,40 However, vesicles formed by these amphiphiles were
observed to be unstable in concentrated solution, in the presence
of salt, and with a pH change. In order to prepare pH-responsive
stable vesicles, we have studied the self-assembly properties of
SOBH in aqueous solution. The major objectives of this study
are (i) to characterize the aggregates formed by SOBH in
aqueous solution, (ii) to investigate the effect of pH, salt
concentration, and temperature on the self-assembly formation
of SOBH, and (iii) to examine vesicle and chiral aggregate
formation in aqueous solution. In this report, we also present the
results of physical changes in membranes induced by mixing of
SOBH with cholesterol by fluorescence anisotropy using 1,6-
diphenylhexatriene as the probe molecule. We have used various
techniques, such as surface tension, conductivity, fluorescence,
dynamic light scattering, confocal fluorescence microscopy
(CFM), and transmission electron microscopy (TEM) to char-
acterize the system.
Identification of chemical structures of SOBH
Yield 65%, M.P. 145–147 C (d). [a]2D5 (0.5%, CH3OH) ¼ 6.80ꢁ,
ꢁ
FT-IR (KBr, cmꢀ1): 3398,3304,3155, 2925, 2853, 1718, 1637,
1
1538, 1508,1400,1313,1250. H-NMR: dH (400 MHz, CD3OD):
7.7 (2H, d, J8.8, Ph-H), 6.9 (2H, d, J8.8, Ph-H), 7.95 (1H, d, J8.8,
NH), 8.49 (1H, S, CCHN), 7.2 (1H, S, NCHN), 4.72 (1H, dd,
J15.2, J25.2, CH(NH)COOH), 4.0 (2H, t, J6.5, O–CH2), 3.33 (2H,
dd,J7.6 CHCH2), 1.8 (2H, m, CH2-CH2–O), 1.47(2H, m, CH2-
CH2-CH2–O),1.32 (8H, m, alkyl chain), 0.9 (3H, t, J6.4, CH3).
13C-NMR: dC (400 MHz, CD3OD) 174.6 (COOH), 167.6
(CONH), 162.1, 128.7, 116.7, 113.6 (Ph-C), 133.3,131.3,125.6
(Im. ring -C), 67.7 (CH2OPh), 53.7, 31.5 (valine C), 29.0, 28.9,
28.8, 27.9, 25.6, 22.2, 12.9 (R).
Methods and instrumentation
The FT-IR spectra were measured with a Perkin-Elmer (Model
Spectrum Rx I) spectrometer. The 1H NMR spectra were
recorded on a Bruker 400 MHz instrument. The melting point
measurement was done using Instind (Kolkata) melting point
apparatus with open capillaries. A digital pH meter (Model PH
5652, EC India Ltd, Kolkata) was used for pH measurements.
Conductivity was measured with a Thermo Orion conductivity
meter (model 150 A+) by use of a cell having a cell constant equal
to 0.467 cmꢀ1. The measurements of optical rotations were per-
formed with a JASCO (Model P-1020) digital polarimꢁ eter. All
measurements were done at room temperature (ꢂ30 C) unless
otherwise mentioned.
Experimental section
Materials
4-Hydroxybenzoicacid, 1-bromooctane, and anhydrous potas-
sium carbonate, sodium bicarbonate, N-hydroxysuccinimide
(NHS), 1,3-dicyclohexylcarbodiimide (DCC), L-histidine were
purchased from SRL, Mumbai, India and was used without
further purification. The fluorescence probes 5(6)-carboxy-
fluorescein (CF), pyrene, and 1,6-diphenyl-1,3,5-hexatriene
(DPH) were obtained from Aldrich (Milwaukee, WI, USA) and
were purified by repeated recrystallization from an ethanol-
acetone mixture. Cholesterol was also obtained from Aldrich.
Analytical grade sodium hydroxide, and hydrochloric acid were
procured locally and were used directly from the bottle. All the
organic solvents were of good quality and commercially
available and were dried and distilled fresh before use. Double
distilled water was used for the preparation of aqueous
solutions.
Surface tension measurements were carried out with a surface
€
tensiometer (model 3S, GBX, France) using the Du Nuoy ring
detachment method. The Pt–Ir ring was carefully cleaned with
50% ethanol–HCl solution and finally with distilled water. The
ring was carefully flamed before measurement. The glassware
was thoroughly cleaned with sulfochromic acid and water. The
instrument was calibrated and checked by measuring the surface
tension of distilled water. A stock solution of surfactant was
made in double distilled water. An aliquot of this solution was
transferred to a beaker containing a known volume of buffer
solution. The solution was gently stirred magnetically through
a magnetic stirrer attached with the machine and allowed to
stand for about 5 min. The temperature of the solution was
controlled at 30 ꢁC by a thermostat, and then surface tension was
measured. For each measurement at least three readings were
taken and the mean g value was recorded.
Synthesis
N-[4-n-octyloxybenzoyl]-L-histidine was synthesized according
to the procedure described elsewhere.41,42 Briefly, 4-octyloxy-
benzoic acid was first synthesized from 4-hydroxybenzoic acid
and 1-bromooctane and purified according to the reported
procedure.43 The coupling of L-histidine and 4-octyloxybenzoic
acid was made via the formation of the NHS ester in the presence
of DCC in THF–H2O medium. The product was precipitated out
from aqueous solution at around pH 4–5 and, the compound was
purified by recrystallization from the ethanol–water mixture. The
In the absence of cholesterol vesicles were formed spontane-
ously when SOBH was dissolved in different pH. Separate
solutions of SOBH in the presence of varying amount of
cholesterol were made by adding different mole ratios of the
surfactant (SOBH) in methanol and cholesterol in chloroform
and were evaporated to dryness in glass vials under a stream of
dry nitrogen gas and were kept under high vacuum overnight for
complete removal of the last trace of organic solvent. The
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surfactant/cholesterol mixtures were dispersed in a phosphate
buffer of appropriate pH for 4 h at room temperature. Finally
the dispersion thus obtained was sonicated individually for 2–5
min to give the resulting solution.
decay of the autocorrelation function of the concentration fluc-
tuations has a characteristic decay rate, G, which is proportional
to q2 (G ¼ Dq2) that characterizes a translational diffusion with
the mutual diffusion constant, D. The scattering vector, q, is
given by the equation:
The UV-visible spectra were recorded on a JASCO (Model
V-530, Japan) spectrophotometer. Fluorescence spectra of the
pyrene probe (ꢂ2 ꢃ 10ꢀ7 M) were measured with a SPEX
Fluorolog-3 spectrofluorometer. The samples were excited at
335 nm and the emission spectrum was recorded between 350
and 450 nm. The steady-state fluorescence anisotropy of DPH
was measured on a Perkin Elmer LS-55 luminescence spec-
trometer equipped with filter polarizers that used the L-format
configuration. The software supplied by the manufacturer
automatically determined the correction factor and anisotropy
value. The r-value was calculated employing the following
equation:
q ¼ 4pn/l0sin(q/2)
(2)
where n is the refractive index of the solvent, and q is the scat-
tering angle. The measured translational diffusion constant was
related to the average hydrodynamic radius, RH, of the particles
through the equation:
D ¼ kT/6phRH
(3)
where k is the Boltzmann constant, h the solvent viscosity, and T
the absolute temperature.
The samples for high resolution transmission electron micro-
scopic measurements were prepared according to the usual
procedure, described as follows. A 5 mL of the surfactant solution
was placed on the 400 mesh carbon-coated copper grid, blotted
with filter paper, and negatively stained with freshly prepared
1.5% phosphotungstate after adjustment of pH to the solution
pH. The specimens were dried overnight in desiccators before
measurement with a high resolution transmission electron
microscope, HRTEM (JEOL-JEM 2100, Japan) operating at
200 kV.
r ¼ (IVV ꢀ GIVH)/(IVV + 2GIVH
)
(1)
where IVV and IVH are the fluorescence intensities polarized
parallel and perpendicular to the excitation light, and G ( ¼ IHV
/
IHH) is the instrumental grating factor. In all cases, the anisot-
ropy values were averaged over an integration time of 10 s and
a maximum number of five measurements for each sample. The
temperature of the water-jacketed cell holder was controlled by
use of a Thermo Neslab RTE 7 circulating bath. Since DPH is
insoluble in water, a 0.5 mM stock solution of the probe in
methanol was prepared. The final concentration of the probe was
adjusted to 1.0 mM by addition of an appropriate amount of the
stock solution. The sample was excited at 350 nm and the
emission intensity was followed at 450 nm using excitation and
emission slits with band-pass of 2.5 and 2.5–7.0 nm, respectively.
A 430 nm emission cut-off filter was used to reduce scattered and
stray radiation. All fluorescence measurements were carried out
at 30 ꢄ 0.1 ꢁC. The measurements started 2–3 h after sample
preparation.
The optical light micrographs for the samples were obtained
from a Leica-DM4500 optical microscope. Confocal fluores-
cence microscopic images were obtained with a FV 1000
Olympus Confocal Microscope with PLAPON 60 X oil immer-
sion objectives. The numerical aperture was 1.42, a 488 nm laser
was used as the excitation source. The fluorescence was viewed
using a 520 nm filter. The image was processed using FV10-ASW
1.6 viewer software. For fluorescence microscopy, CF was
trapped into the vesicle by gentle mixing of the surfactant (1 mM
for pH 8 and 8 mM for pH 12) and CF (1 mM) into methanol
and followed by rotary evaporation. The dry film thus produced
was soaked overnight in buffer of appropriate pH, vortexed and
sonicated for 5 min. The aqueous solution was obtained after
appropriate dilution. The excess dye was removed by dialysis for
10–12 h using an ultrafiltration cellulose acetate membrane bag
(16 mm diameter, 10 kDa MWCO). An aliquot of the undiluted
vesicle solution was pipetted onto the glass slide and sealed with
a coverslip and left to sit the coverslip down for a few minutes
before analysis.
Time-resolved fluorescence measurements were performed on
a time-correlated single-photon counting method that uses
a picosecond diode laser at 370 nm (IBH, U.K., nanoLED-07)
as a light source. The typical response time of this laser system
was 70 ps. Fluorescence lifetimes were determined from time-
resolved intensity decay by the method of time-correlated single-
photon counting. The decays were analyzed using IBH DAS-6
decay analysis software. For all the lifetime measurements the
fluorescence decay curves were analyzed by a biexponential or
triexponential iterative fitting program provided by IBH.
The dynamic light scattering (DLS) measurements were per-
formed with a Zetasizer Nano ZS (Malvern Instrument Lab,
Malvern, U.K.) optical system equipped with a He–Ne laser
operated at 4 mW at lo ¼ 633 nm, and a digital correlator. Before
measurement, the scattering cell was rinsed several times with the
filtered solution. The DLS measurements started 5–10 min after
the sample solutions were placed in the DLS optical system to
allow the sample to equilibrate at room temperature. ꢁFor all
light-scattering measurements, the temperature was 30 C. The
scattering intensity was measured at a 173ꢁ angle to the incident
beam. The data acquisition was carried out for 10 min and each
experiment was repeated two or three times. The data were
analyzed using the second-order cumulant method.44 The time
Results and discussion
Ionization behavior in solution
To our surprise, it was found that the amphiphile (SOBH) has
very low solubility in water in the pH range 3–7 at room
temperature. The pKa values corresponding to the protonation
of COOꢀ and dissociation of -NH+ imidazole groups for histidine
are 1.77 and 6.1, respectively.45 But for long chain amphiphiles
the pKa values are usually higher.46 Indeed, the pKa values of
Na-octadecanoyl-L-histidine at 25 ꢁC, as reported by Kashima
et al. are 3.12 (COOH) and 7.49 (NH+).46b Thus, SOBH is also
expected to have similar pKa values. Consequently, it was
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Table 1 The self-assembly properties of SOBH in 20 mM phosphate
buffer of pH 8.0 and pH 12.0 at 30 ꢁC
Properties of SOBH
pH ¼ 8.0
pH ¼ 12.0
CAC (mM)
gCAC/mN mꢀ1
pC20
0.26
31.0
4.42
2.0
41.0
0.66
1.11
55.8
0.84
36.4
3.90
1.83
45.0
0.60
1.38
46.2 (22.3)a
Gmax ꢃ 106 (mol mꢀ2
)
2
Amin (A molecule
ꢀ1
ꢀ
)
P
I1/I3
hm (mPa s)
a
The value within the parentheses was obtained using 8.0 mM SOBH at
pH 12.0.
parameters, such as CAC, surface tension corresponding to CAC
(gCAC), and efficiency of adsorption, pC20 (¼ negative logarithm
of surfactant concentration required to reduce the g of water by
20 units) were determined from the ST plot for SOBH at pH 8.0
and 12.0. All the physicochemical parameters are listed in Table 1.
The values of the surface excess (Gmax) and cross-sectional area
per head-group (Amin) at the air/water interface were calculated
by using the Gibbs adsorption equations,47,48
Scheme 1 The proton transfer equilibria in aqueous solution of SOBH.
observed to become soluble in water at pH>7 as it transformed
into the anionic form. But below pH 3.0 it forms a turbid solu-
tion. Therefore, the aggregation behavior of the amphiphiles was
studied at pH 8.0 and 12 where it exists mostly in the zwitterionic
and anionic form, respectively, as shown in Scheme 1.
Gmax ¼ ꢀ1/2.303nRT (dg/dlogC)
Amin ¼ 1/NA Gmax
(4)
(5)
Surface tension studies
where dg/dlogC is the maximum slope; NA is Avogadro’s
number; T ¼ absolute temperature; n ¼ 1 for a 1 : 1 ionic
surfactant in the presence of a swamping amount of 1 : 1 elec-
trolyte;47 R ¼ 8.314 J molꢀ1 Kꢀ1. The cross-sectional areas were
calculated from Gmax values corresponding to the break of the ST
plot (Fig. 1). It should be noted that SOBH has a slightly lower
Amin value (Table 1) at pH 8.0 than at pH 12.0. The smaller value
of Amin at both pH suggests the formation of large aggregates
with tightly packed hydrocarbon chains. This is further indicated
by the critical packing parameter, P ( ¼ v/Aol, where Ao is the
area per headgroup in the aggregate, v and l are the volume and
length of the hydrophobic chain, respectively)49 of the species at
the above pH values. With reasonable accuracy, P can be
equated to v/Aminl.50 The value of v (0.419 nm3) was calculated
using the atomic increment method after appropriate correc-
tion.51 On the other hand, the value of l (1.54 nm) was calculated
as the fully stretched chain of the energy minimized (MM2)
structure using Chem3D (version 5.0) program. The P values
thus obtained have been included in Table 1. The large values of
P (>0.5) at both pH are indicative of the existence of bilayer
structure.49 The formation of spherical bilayer vesicles for this
class of amphiphiles in dilute solution is due to intermolecular
hydrogen bonding (IHB) between amide groups and p–p
stacking interaction between benzene rings and the close packing
of the hydrocarbon tails which has been reported earlier3,40,42 and
has been substantiated by the results of fluorescence probe
studies discussed below.
The critical aggregation concentration (CAC) of the chiral
surfactants was measured by the surface tension (ST) method,
which is versatile not only because CAC can be extracted from
the plots of surface tension (g) versus log[Surfactant] but also
information can be drawn on the nature of adsorbed layers at the
air/water interface. The typical plots of surface tension versus log
C (logarithmic value of surfactant concentration) for an aqueous
solution at pH 8.0 and 12.0 of the surfactants are shown in Fig. 1.
The surface tension values decrease linearly with log C and show
a characteristic break and remain constant thereafter. In pH 8.0
the plot shows a hump, which represents the presence of two
forms of molecule, one is in anionic form and another in
zwitterionic form. The concentration corresponding to the
breakpoint was taken as the CAC value. The surface-active
We have also measured the CAC value of SOBH in different
pH solutions using the ST method. Variation of CAC as a func-
tion of pH is shown by the plot in Fig. 2. It can be found that
CAC increases with pH reaching a plateau at pH>11, which must
Fig. 1 A plot of surface tension (g) vs. log C of SOBH in 20 mM
phosphate buffer at pH 8.0 and 12.0 at 30 ꢁC.
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negatively charged headgroups, which favors the formation of
bilayer aggregates in dilute solution. Consequently, the packing
of the hydrocarbon chains becomes less tight compared to the
bilayer aggregates in pH 8.0, which allows more water molecules
to penetrate into the hydrocarbon core of the aggregate. This
means an increase of both fluidity and polarity of the microen-
vironments of probe molecules.
In order to quantify the rigidity of the hydrocarbon chain of
the aggregates formed in pH 8.0 and 12.0, we have determined
microviscosity around the DPH probe in the surfactant aggre-
gate. The microviscosity (hm) can be calculated from the Debye-
Stokes-Einstein relation54,55 using the r-value and fluorescence
lifetime (sf) of the DPH molecule in the presence of surfactant
according to the procedure described in the literature.56 The sf
values obtained from the analysis of fluorescence intensity decays
of the DPH probe in the presence of the SOBH amphiphile at pH
8.0 and 12.0 are 5.39 ns, and 5.3 ns, respectively. The micro-
viscosity values thus calculated are presented in Table 1. The
hm-value for the self-assemblies of SOBH at pH 12.0 at higher
concentration (8.0 mM) is 22.32 mPa s, which is similar to those
of micellar aggregates of SDS and DTAB surfactants.56
However, at low surfactant concentration, the hm-value for the
self-assemblies at pH 8.0 (55.8 mPa s) and 12.0 (46.2 mPa s) are
larger than that of ionic micelles.56 The relatively higher hm-value
in a dilute solution of SOBH is consistent with the formation of
bilayer aggregates. The lower values of hm for the bilayer
aggregates compared to liposomes is due to quenching (colli-
sional) of fluorescence of the DPH molecule by the imidazole
ring present in the head group of SOBH, which reduces the sf
value. This was verified by a simple experiment. We made two
solutions of sodium N-(4-n-octyloxybenzoyl)-L-valinate (SOBV)
and SOBH having the same DPH and surfactant concentration.
It was observed that the fluorescence intensity of DPH was quite
low for the SOBH surfactant in comparison to SOBV3 at room
temperature. It should also be noted that the sf value of the DPH
probe in SOBH (5.39 ns) is less than that observed in the SOBV
(6.30 ns) solution. In fact, the ratio (1.14) of fluorescence inten-
sity in SOBV to that in SOBH is almost equal to the ratio (1.16)
of the corresponding fluorescence lifetimes, which suggests that
the fluorescence quenching in the SOBH solution is dynamic in
nature.
Fig. 2 The variation of the CAC of SOBH with pH in 20 mM phosphate
buffer at 30 ꢁC.
be due to the gradual conversion of the zwitterionic species to
anionic species at higher pH. Increased ionic repulsion among
surfactant headgroups at pH>11 decreases hydrophobic inter-
action and thus increases CAC value. The pH (ca. 8.2) corre-
sponding to the inflection point of the curve can be taken as the
macroscopic pKa of the NH+ group of the imidazole ring. It is
interesting to note that the pKa of the NH+ group of SOBH is
higher than that of Na-octadecanoyl-L-histidine.46(b)
Fluorescence probe studies
In order to understand the pH dependent structural change of
the surfactant aggregates, we have performed steady-state fluo-
rescence anisotropy (r) measurements. The r is an index of
equivalent microviscosity in the vesicle lipidic core. The DPH is
a well-known membrane fluidity probe.52 Therefore, the r-value
of DPH was measured in different buffer solutions in the pH-
range of 8–12. The r-value is relatively high for lower pH (ꢂ8.0),
which decreases with an increase in pH as shown by the plot in
Fig. 3. The relatively high values of r in pH 8.0 and 12.0 suggest
an ordered environment around the DPH probe in the self-
assemblies and thus supports the existence of bilayer
structures.3,40,42,53 The results are thus consistent with those
obtained by ST studies. Slightly lower values of r in pH 12.0
(ꢂ0.16) is due to the electrostatic repulsion among headgroups in
the aggregate that weakens the hydrophobic interaction of the
hydrocarbon chains. However, the presence of the bulky imid-
azole ring reduces the electrostatic repulsion among the
For bilayer aggregates, as discussed before, the packing of the
hydrocarbon chains of the amphiphilic molecules in the aggre-
gate is very tight compared to micellar aggregates (e.g. spherical
or rod-like micelles). This means that the hydrocarbon region of
the bilayer aggregates will be much less polar compared to
micellar aggregates. The micropolarity of the self-assemblies
formed in pH 8 and 12 was therefore measured by use of a pyrene
probe whose fluorescence property is sensitive to polarity change.
It is well known that the ratio of the intensities of the first and
third vibronic bands (I1/I3) in the fluorescence spectrum of
pyrene is sensitive to solvent polarity change.57,58 Therefore, this
parameter was used to test changes in the micropolarity of the
aggregates upon change of solution pH. The data are plotted
against pH in Fig. 3. The I1/I3 value for the SOBH amphiphile
measured at a concentration around two times its CAC value is
much less than that in the pH 12.0 buffer (1.80), indicating
solubilization of the pyrene molecule in the hydrophobic region
of the aggregate. However, it is interesting to observe that the
Fig. 3 A plot of r and I1/I3 against pH in the presence of 1.5 mM SOBH
at 30 ꢁC.
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I1/I3 index decreases further with the decrease of pH (Fig. 3)
indicating a decrease of micropolarity. This is consistent with the
decrease of membrane rigidity (hm) of the bilayer self-assembly
with the increase of pH as indicated by the decrease of fluores-
cence anisotropy (r) of DPH with the increase of pH. This must
be a consequence of the transformations of SOBH from zwit-
terionic to anionic form which results in an increase of electro-
static repulsion among headgroups, thus allowing penetration of
more water molecules into the bilayer and thereby increaseing
the micropolarity parameter. This means leakage of entrapped
drugs, if any, can be facilitated by increase of the pH. In other
words, controlled release of drugs can be achieved.
It is important to note that the pKa value of the imidazole
nitrogen as obtained from the titration curves in Fig. 3 is ca. 9.5
which is higher than the macroscopic pKa (ꢂ8.2) value of SOBH.
This must be due to aggregate formation. Such aggregation
induced pKa shift has also been noted by others.59 The higher pKa
value for the long chain amphiphilic molecule is because the local
pH value at the surface of the negatively charged aggregate is
lower than the pH of the bulk solution.
Fig. 5 The characteristic specific conductance change (Dk) vs. [SOBH] in
the presence of 3 mM KCl in phosphate buffer of pH 12.0 at 30 ꢁC.
of salt solution upon addition of vesicle-forming surfactants has
also been reported by others.60 The conductivity of a 3 mM KCl
in the presence of different concentrations of the surfactant was
measured at 30 ꢁC. It was observed that the sum of the
conductivities of the solutions of 3 mM KCl and 1 mM SOBH
(20 mM phosphate buffer pH 12.0) was greater than that of
1 mM SOBH (20 mM phosphate buffer pH 12.0) containing
3 mM KCl. This means that the conductivity of the KCl solution
is decreased in the presence of 1 mM (>CAC1) SOBH. The
variation of conductivity change (Dk) with the increase of
[SOBH] is shown in Fig. 5. It can be observed that although the
initial value of Dk is small, it increases nonlinearly with surfac-
tant concentration. The Dk value reaches a maximum at around
4 mM and then drops down at [SOBH]>4 mM. This is clear
evidence of the formation of vesicles that have an aqueous core in
concentrations up to 4 mM in pH 12.0. Since both size and
population of vesicles increases with surfactant concentration,
more K+ and Clꢀ ions get trapped inside the aqueous core and
hence display an increase in the Dk value. The fall of the Dk value
above 4 mM is perhaps due to transformation of vesicles to other
kind of aggregate structures that lack aqueous core like vesicles.61
Indeed as suggested by the concentration dependence of fluo-
rescence anisotropy of the DPH probe the vesicles are trans-
formed into rod-like micelles. That the release of the entrapped
water and ions occurs at a high surfactant concentration is
supported by the lowering of the fluorescence anisotropy of the
DPH probe (see Fig. 4).
In order to investigate the concentration dependent aggrega-
tion behavior of SOBH, we also measured the fluorescence
anisotropy of the DPH probe at different concentrations in pH
12. The result is presented in Fig. 4, which shows that the r-value
continuously decreases with the increase of [SOBH] up to 12 mM
and thereafter remains unchanged. In light of the above discus-
sion, the low r-value in concentrated solution can be associated
with the formation of rod-like micelles. Such studies in pH 8
could not be done at room temperature because of precipitation
of SOBH at concentrations higher than 1.0 mM. Due to strong
electrostatic attraction the large aggregates that are formed at
higher concentration fall out of solution.
Conductivity measurements
To examine the shape of the bilayer structures formed in pH 12
buffers we performed conductivity measurements. Closed vesic-
ular structures can be confirmed from conductivity measure-
ments of the vesicular solution in the presence of KCl. Bilayer
aggregates, such as vesicles, entrap a part of the water and the
salt present in it. Consequently the entrapped charge carrier ions
are prevented from contributing to the conductivity of the
solution, which means a decrease of the conductivity of the
vesicular solution. In fact, the decrease of electrical conductivity
Transmission electron microscopy (TEM)
To further visualize the shape of the surfactant aggregates in
solution we obtained TEM pictures of negatively stained samples
of the dilute solutions of SOBH at pH 8.0 and pH 12.0 (Fig. 6(a)–
(c)). The images in Fig. 6(a) and 6(b) clearly reveal the existence
of spherical vesicles with an aqueous cavity. This supports the
results obtained from the ST, fluorescence, and conductivity
studies. The inner diameters of the vesicles are in the range 30–
100 nm. The formation of smaller size vesicles of around 30–50
nm diameter in dilute aqueous solutions of pH 8 is due to the
presence of the mostly zwitterionic form of the amphiphiles. The
image in Fig. 6(c) taken for a 10 mM solution in pH 12.0 reveals
a bunch with rod-like morphology. These are a few micrometres
long and have very small inner diameter. The transition from
Fig. 4 A plot of r of DPH against [SOBH] in pH 12.0 buffer at 30 ꢁC.
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Fig. 6 Negatively stained (sodium phosphotungstate) transmission electron microscopic images of aqueous SOBH: (a) 0.5 mM, pH 8.0, (b) 2.0 mM, pH
12.0, and (c) 10 mM, pH 12.
vesicle to rod-like micelles at higher surfactant concentration has
been previously reported by us.62 The transformation of vesicles
to rod-like structures is consistent with the decrease in fluores-
cence anisotropy (see Fig. 4) of the DPH probe and specific
conductance (see Fig. 5) of KCl salt with the rise of surfactant
concentration above 4 mM. In fact, the appearance of fiber-like
aggregates was observed upon standing of the sample for a few
days. This can also be visualized in the optical micrographs
shown in Fig. S2 of the ESI†. The micrograph (b) of Fig. S2† also
reveals the existence of helical aggregates.
measurements using dilute aqueous solutions of SOBH. The
optical images (Fig. 7) obtained for samples in pH 8 and 12
exhibit vesicular structures. However, only large vesicles could be
seen under the microscope. The smaller vesicles having a diam-
eter of less than 100 nm could not be observed because of limited
resolution of the microscope. The existence of vesicles in dilute
solutions of both pHs is thus confirmed.
Hydrodynamic size of the aggregates
The mean hydrodynamic diameter (dH) of the aggregates was
measured by DLS technique at different concentrations and pH.
The volume distribution graphs for the amphiphile are shown in
Fig. 8. It is observed that the aggregates formed in pH 8 and 12
have narrow and bimodal size distributions. The aggregates are
found to be much larger in size than small spherical micelles
which have diameters typically in the range 3–5 nm.63 The dH
values of the aggregates in 0.5 mM (pH 8) are found to be in the
range of 30–200 nm, whereas those in 2.0 mM (pH 12) solution of
SOBH the particle size is in the range 60–300 nm. In both pHs,
the volume fraction of the larger size particles is much lower
compared to the small size particles. However, the sizes of the
small and large particles are closely equal to the vesicle structures
observed in the corresponding TEM as well as optical micro-
graphs. At higher SOBH concentration (8.0 mM, pH 12), the
z-average dH value is ca. 1.0 mm (this value is not correct because
the principle involved in DLS measurements is only applicable
for spherical particles) indicating the existence of large aggre-
gates. This is consistent with the TEM picture (Fig. 6(c)) and can
be attributed to the transformation of the small spherical vesicles
to large rod-like micelles upon increase of surfactant concen-
tration. The appearance of a small hump on the higher size range
of the distribution in Fig. 8(c) perhaps indicates that large fibers
separate out of solution with time. Indeed, the appearance of
long fibrous aggregates could be observed even with naked eye
when the concentration was increased further.
Confocal fluorescence microscopy
To further support the results obtained from TEM studies we
have also performed confocal fluorescence microscopic
Influence of salt concentration
The influence of salt concentration and temperature on the
stability of vesicle was investigated by monitoring the fluores-
cence anisotropy (r) of the DPH probe in surfactant solutions. In
the presence of NaCl salt the r-value was observed to decrease.
Fig. 9 shows the plot of the variation of r as a function of [NaCl]
for a particular concentration of SOBH. It is observed that for
Fig. 7 Fluorescence microscopic images of aqueous SOBH: (a) 0.5 mM,
pH 8.0, and (b) 2.0 mM, pH 12.0.
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Fig. 8 The size distribution profile of aggregates in (a) 0.5 mM, pH 8.0, (b) 2.0 mM, pH 12, and (c) 10 mM, pH 12 SOBH solution.
Fig. 10 A plot of fluorescence anisotropy (r) of DPH in the presence of
Fig. 9 A plot of fluorescence anisotropy (r) of DPH in the presence of
0.5 (pH 8) and 2.0 mM (pH 12) SOBH against temperature.
0.5 (pH 8) and 2.0 mM (pH 12) SOBH against [NaCl].
of temperature can be associated with the weakening of the
hydrophobic interactions among hydrocarbon chains in the
aggregate as a result of disruption of the intermolecular
hydrogen bonds and other physical forces. This means that the
hydrocarbon chains become more fluid at higher temperatures.
In other words, a phase transition from a more ordered gel-like
bilayer state to a slightly less ordered liquid-crystal state occurs in
the pH 8 vesicle solution upon increase of temperature. However,
in the pH 12 solution, the r-values in the initial and final states
suggest that the phase transition occurs between vesicle and
micellar states. Since the r-value decreases with the rise in
temperature, the temperature corresponding to the inflection
point can be taken as the melting or phase transition tempera-
ture, Tm of the vesicles. The high melting temperature, (ꢂ40 ꢁC)
for both pH 8 and 12 clearly suggests that the vesicles are quite
pH 8.0, the r-value initially increases, reaches a maximum at ca.
20 mM NaCl and then decreases with a further increase in salt
concentration. But at pH 12.0, a sharp decrease of r-value can be
observed, suggesting the transformation of the bilayer structure
to rod-like micelles at a relatively low NaCl concentration (ꢂ20
mM). Similar behavior was also observed with a SOBV surfac-
tant.3 For the pH 8 solution, even in the presence of 200 mM
NaCl, the r-value is not as low as it is observed with the pH 12
solution. The initial increase in r-value is due to the counter ion
balance that facilitates growth of vesicles. The results indicate
that the vesicles are quite stable under physiological conditions
(pH 7.4, 150 mM NaCl). However, the decrease of membrane
rigidity at higher salt concentrations suggests that NaCl can be
used to induce slow release of encapsulated drugs.
ꢁ
stable at physiological temperature (37 C).
Effect of temperature
It has been found that vesicles formed in different pH have
different physical properties and stability.10,64 For this we have
also studied the stability of vesicles at different temperature in the
range 20–60 ꢁC. The temperature stability of vesicles was studied
by use of a DPH probe. Fig. 10 shows the plots of the variation of
r-value of DPH as a function of temperature for both pH 8 and
12 at a particular concentration of SOBH. The r-value is higher
at low temperature, but it decreases with the rise in temperature.
However, at 60 ꢁC the r-value is much less for pH 12 and
corresponds to micellar structure. On the other hand, for vesicle
solutions at pH 8, the r-value is higher even at 60 ꢁC and indicates
the existence of vesicles. The decrease of r-value with the increase
Effects of cholesterol
Cholesterol (Chol) is known to impart rigidity to phospholipid
membranes and thus control the permeability of cellular
substances through membranes.65 In order to study the interac-
tion of Chol with the bilayer membranes formed by SOBH, we
investigated the effect of adding increasing levels of Chol to
SOBH solution at pH 8.0 and 12.0 and also the variation of
SOBH concentration at fixed mole % of Chol through fluores-
cence anisotropy of the DPH molecule (Fig. 11(a) and (b)).
Usually, there is an upper limit to Chol incorporation in lipid
bilayers, above which Chol seems to precipitate as crystals of
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Fig. 12 A plot of fluorescence anisotropy of DPH versus (a) temperature
and (b) [NaCl] in the presence of 0.5 and 2.0 mM SOBH containing 10
mol % Chol at pH 8.0 and pH 12.0, respectively.
Fig. 11 A plot of the fluorescence anisotropy of DPH (a) as a function of
mol% of Chol in 0.5 mM and 2.0 mM SOBH at pH 8.0 and 12.0,
respectively, (b) against [SOBH] containing 10 mol% Chol at pH 12.0 at
30 ꢁC.
temperature in the lipid bilayer region of the membrane and also
influences their permeability and stability to the external envi-
ronment. In order to assess such behavior we have studied the
effect of temperature, salt, and pH for vesicles formed from
a SOBH-Chol (10%) mixture. The addition of 10 mol% Chol to
lipids generally causes broadening of the phase transition process
and hence distinct thermal phase transitions are not observed.
The temperature effect on the vesicle stability formed from the
SOBH-Chol (10%) mixture has been shown by the plots
(Fig. 12(a)) of the r-value of the DPH probe against temperature.
Although for both pH solutions the anisotropy decreaꢁses with
temperature, the anisotropy values are high even at 60 C, sug-
gesting the existence of a bilayer structure beyond physiological
temperature. The decrease of r-value in the studied temperature
range can be attributed to the gel-to-liquid-crystal phase transi-
tion of the lipid bilayer membranes. The phase transition
temperatures for the SOBH-Chol mixture were obtained from
the inflection point of the plots. Tm values of ca. 37 ꢁC were
obtained for vesicle solutions at both pH 8.0 and 12.0. This
suggests that the packing of hydrocarbon chains of the bilayer
become less rigid and thus can enhance the leaking of encapsu-
lated drug molecules, if any. In other words, the prepared vesicles
may be used as temperature-responsive drug delivery vehicles.
Upon addition of the Chol in the lipid matrix, the Chol
molecules are incorporated into the bilayer resulting in a greater
headgroup separation between the lipid monomers. Conse-
quently, incorporation of Chol into lipids offers the vesicles
a greater stabilization as a result of the minimization of head-
group-headgroup repulsions relative to that present in pure
surfactant assemblies. Such stabilization can protect the vesicular
structure from external physical factors, such as pH and salt
(NaCl). We also found that with increasing NaCl concentration
the fluorescence anisotropy exhibits very little change
(Fig. 12(b)). High anisotropy value in the presence of 200 mM
NaCl suggests that the vesicles formed with SOBH-Chol mixture
are more stable than those formed by SOBH only. Similarly,
keeping the molar concentration of the SOBH-Chol mixture
fixed the solution pH was varied in the range 8–12. However, no
significant change in r-value was observed with the decrease of
pH (results are not shown here), indicating higher pH-stability.
This is different from the results obtained in the absence of Chol
(Fig. 3). In the absence of Chol, however, more changes in
r-value were observed. This indicates that pH-induced leaking of
encapsulated drug can be reduced by adding Chol.
pure Chol either in the monohydrate or in the anhydrous form.66
Huang et al.67 observed that for a membrane composed by
phosphatidylcholine (PC) the maximum cholesterol solubility is
about 66 mol% and by phosphatidylethanolamine (PE) the limit
is about 51 mol%, depending on the acyl chain composition. As
the concentration of Chol increases, the system undergoes
a process of phase separation to form Chol-rich domains.68 In
our case, addition of ca.10 mol % Chol gave the highest fluo-
rescence anisotropy value but at still higher mol% Chol, the
r-value decreased. Interestingly, the decreases in anisotropy
observed at the higher levels of Chol appeared to be accompa-
nied by an increase in vesicle aggregation due to less solubiliza-
tion of higher mol % Chol as evidenced by the appearance of
turbidity. The growth of vesicles to larger sizes (perhaps due to
the formation of multilamellar vesicles) is evidenced by the
increase of mean hydrodynamic diameter (see size distributions
in Fig. S2 of the ESI†). However, the latter effect may be due to
the formation of Chol clusters at high Chol content where excess
amounts of Chol cannot interact with the SOBH, and deposits on
the bilayers.
We also found that the presence of 10 mol% Chol with
increasing surfactant concentration at pH 12.0 keeps the
anisotropy values higher than that of the pure surfactant solu-
tion. These anisotropy data provide some quantitative estimates
on the fluidity of the interior of lipid vesicles and lipid-Chol co-
aggregates. Results show that addition of Chol makes the bilayer
membrane more rigid both in pH 8.0 and 12.0. This rigidity
offers more stability to the vesicles, even at a higher concentra-
tion of surfactant, than the vesicles produced by pure SOBH. The
plot in Fig. 11(b) shows a decrease of r-value with [SOBH] in
SOBH-Chol mixture suggesting
a concentration-dependent
phase change in pH 12.0 even in the presence of 10 mol% Chol.
However, a relatively large r-value at higher concentration
indicates that the bilayer structure is still retained. The decrease
of r may therefore be attributed to the transformation of closed
bilayer vesicles to a flat lamellar structure which is evidenced by
the large increase in the hydrodynamic diameter of the aggre-
gates (see Figure S2 of the ESI†).
It is known that the presence of Chol is responsible for the
orientation and packing of the lipid hydrophobic chain.69 This
hydrophobic association of the lipid and Chol imposes a restric-
tion on the chain (CH2)n motions,70 and offers rigidity to the self-
assembly. This in turn alters the thermotropic phase transition
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In summary, we have synthesized a new surfactant, SOBH,
which has very low CAC and gCAC values and acts as a very good
surface-active agent. The results of surface tension, conductivity,
fluorescence, DLS and TEM studies have shown the formation
of vesicular structures by the zwitterionic and anionic forms of
SOBH in the pH range 8–12. The vesicles formed by SOBH have
hydrodynamic diameters in the range of 30–200 nm in pH 8
which increased to 60–300 nm in pH 12. In fact, depending upon
the concentration of SOBH, the pH of the medium and the
presence of salt or cholesterol gives different morphologies of the
organized self-assemblies, for example small spherical vesicles
and ribbons were observed which was suggested by the anisot-
ropy (r) values of the DPH probe, and TEM and optical
micrographs. The spherical vesicles are abundant in dilute
alkaline solutions whereas other bilayer structures are predomi-
nantly present in relatively concentrated solutions and higher
pH. The fluorescence probe studies indicate that the microenvi-
ronment of the self-assemblies is nonpolar and is very viscous,
indicating rigid bilayer membranes, but they are capable of
encapsulating polar molecules ,which was suggested by
a conductivity experiment using KCl in the presence of SOBH.
The vesicles produced in pH 8 solution are more stable than
those formed in pH 12. The presence of cholesterol increases the
rigidity and offers stability to the bilayer structures due to
hydrophobic association with the alkyl chain of the amphiphile.
The studies indicated that the vesicles obtained from pure SOBH
and SOBH-Chol mixture can have potential applications in salt-
and temperature-induced sustained release of drugs.
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Acknowledgements
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The authors gratefully acknowledge financial support for the
work (Grant No.SR/S1/PC-18/2005) from the Department of
Science and Technology, New Delhi. TP thanks CSIR (9/
81(594)/2006-EMR-I) for a research fellowship. The authors
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