Comptes Rendus Chimie

Sampad Ghosh

Anirban Ray

Nabakumar Pramanik

Balram Ambade

lable at ScienceDirect
C. R. Chimie 19 (2016) 951e954Contents lists avaiComptes Rendus Chimie
www.sciencedirect.comFull paper/MemoireCan a catanionic surfactant mixture act as a drug delivery
vehicle?
Sampad Ghosh a, *, Anirban Ray b, Nabakumar Pramanik c, Balram Ambade d
a Chemistry Department, V.B. College of Education, Bhagalpur, Bihar, 813210, India
b Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani, West Bengal, 741235, India
c Department of Chemistry, National Institute of Technology, Arunachal Pradesh, 791112, India
d Department of Chemistry, National Institute of Technology, Jamshedpur, 831014, Indiaa r t i c l e i n f o
Article history:
Received 24 December 2015
Accepted 24 March 2016
Available online 8 June 2016
Keywords:
Catanionic mixture
cmc
Dye entrapment
Hemolytic assay
Biocompatibility
Cytotoxicity* Corresponding author.
E-mail address: justsampad@gmail.com (S. Ghos
http://dx.doi.org/10.1016/j.crci.2016.03.020
1631-0748/© 2016 Académie des sciences. Publishea b s t r a c t
Surfactants can self-assemble in dilute aqueous solutions into a variety of microstructures,
including micelles, vesicles, and bilayers. Recently, there has been an increasing interest in
unilamellar vesicles, which are composed of a closed bilayer that separates an inner
aqueous compartment from the outer aqueous environment. This interest is motivated by
their potential to be applied as vehicles for active agents in drug delivery via several routes
of administration. Active drug molecules can be encapsulated in the bilayer membrane if
they are lipophilic or in the core of the vesicle if they are hydrophilic. Furthermore vesicles
formed by mixing of cationic and anionic surfactants (so called ‘catanionic’ systems) can be
used as models for biological membranes as they have low critical micelle concentration
(cmc) and are highly biocompatible. In this work the formation of amino acid based mixed
surfactant vesicles and their stabilization and biocompatibility were studied systematically
using several instrumental techniques.
© 2016 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.1. Introduction
Drug delivery is an interdisciplinary area of research in
which people from almost every scientific discipline can
make a significant contribution. Catanionic vesicles have
already been applied as templates for drug-delivery appli-
cations as well as in nano-drug carrier synthesis [1e5].
Catanionic vesicles can be tailored in size and bi-layer
thickness by changing the characteristics of the two sur-
factants, such as the type of the polar head and chain
length, presence of salt and cholesterol, etc [6]. Owing to
the vesicle structure, they compartmentalize the aqueous
domain in the inner core of the vesicles which is separated
from the outer water by a hydrophobic bi-layer [7]. They
display therefore, interesting features like pH sensitivity ash).
d by Elsevier Masson SAS. Afar as the synthesis of different stable vesicles of finite size
is involved [8]. Greater the sensitivity of the vesicle archi-
tecture in differing pH in the local environment greater is
the use as the template for drug delivery systems. However,
as technology has advanced over the past few decades,
methods of drug delivery became more sophisticated and
rational, and people incite new strategies, including novel
liposomes or vesicles for drug administration.
Attempts have been made to explore the potential ap-
plications of the vesicular systems to act as drug carriers in
pharmaceutical industries. The formation, stability and
biocompatibility studies of the mixed surfactant vesicles
were thus examined in terms of different characterization
techniques, hemolysis and MTT (3-(4,5 dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay.
Although this type of mixed surfactant vesicle applied here
exhibits enhanced stability in the presence of external
stimuli, like salt, cholesterol, pH it is expected to encap-
sulate drugs in its core [6]. The drugs encapsulated in thell rights reserved.
S. Ghosh et al. / C. R. Chimie 19 (2016) 951e954952aqueous core of the vesicles are also stable over a certain
period of time in the physiological pH-temperature ranges,
and can be considered as an effective drug delivery vehicle.
Therefore, the main objective of the present study is to
investigate the vesicle stability, pH-induced release of
model drugs, hemocompatibility and cytotoxicity of the
mixed surfactant vesicles using amino acid based carbox-
ylate surfactants sodium N-alkanoyl-L-sarcosinate (SOS,
SDS, SLS, and STS) of varying chain length and two com-
mercial cationic surfactants cetyltrimethylammonium hy-
droxide (CTAOH) and dodecyltrimethylammonium
hydroxide (DTAOH).
2. Experimental section
2.1. Materials
Sodium N-alkanoyl-L-sarcosinate (SAS) surfactants with
different chain lengths (see Fig.1 for structure) employed in
this work were synthesized according to the reported
methods [9,10]. L-alanine was obtained from SRL (Mumbai,
India) and was used without further purification. The
cationic surfactants, CTAOH and DTAOH, were prepared
from CTAB and DTAB, respectively, by using anionic ion
exchanger resin, where the ion exchange capacity of the
anionic exchanger was fixed at 0.9 mM/ml. The model
drug calcein (a fluorescent dye) was purchased from
Aldrich (Milwaukee, WI, USA) and was used after recrys-
tallization at least three times from an ethanol or aceto-
neeethanol (1:1) mixture. All the reagents and solvents
used in this experiment were of good quality commercially
available and were purified or distilled fresh whenever
required.
2.2. General instrumentation and solution preparation
All the measurements were carried out at room tem-
perature (30 C) unless otherwise mentioned. Temperature
controlled measurements were carried out using a circu-
lating water bath. Samples were prepared by combining
the cationic and anionic surfactants at a desired concen-
tration and mixing ratio from the individual surfactant
stock solution. After sealing the samples were mixed by
simple hand agitation. The composition of the mixtures
thus obtained is expressed in terms of molar fraction, X1
(¼[SAS]/([SAS] þ [CTAOH])) of SAS. The pH of the samples
was constant (pH 7.4) at 20mMphosphate buffer having an
ionic strength of 0.0126 M. Prior to observation and mea-
surements, the solutions were allowed to equilibrate atFig. 1. Chemical structure of sodium N-alkanoyl-L-sarcosinate (SAS).room temperature for a 2e3 h. Transmission electron mi-
croscopy (TEM), dye entrapment and vesicle stability
determination, hemolytic assay and cytotoxicity assay (cell
culture andMTTassay) were performed as per our previous
investigations [11].
The phase behavior of the SAS-CTAOH (or DTAOH)
mixtures at different mixing ratios and concentrations was
first investigated for aqueous solubility. It was observed
that binary mixtures of SAS and CTAOH produced optically
isotropic clear solutions at all compositions and total con-
centrations tested.3. Results and discussion
The catanionic mixture formed by anionic amino acid
based carboxylate surfactants sodium N-alkanoyl-L-alani-
nate (SOS, SDS, SLS, and STS) of varying chain length and
two commercial cationic surfactants DTAOH or CTAOHwith
different chain lengths forms a strong synergistic interac-
tion and stable vesicles in different compositions as dis-
cussed earlier [12]. The presence of vesicles are mostly seen
at X1 ¼ 0.50, whereas in other compositions both micelles
and vesicles are found. The surface tension (also cmc) value
of the mixed system is found to be minimum at different
compositions andmuch lower than the pure and individual
surfactants [13]. The fluorescence anisotropy values are
also much higher (>0.140) in different compositions [11]
which suggest the formation of vesicular systems which
is further supported by the transmission electron micro-
graph (TEM) in Fig. 2. The TEM images show the hollow
sphere structure of the vesicles and the hydrodynamic
diameter (dH) of the vesicles is in the range of ~30e200 nm.
The drug molecules can easily be encapsulated in the
aqueous core of these vesicles. The mixed surfactant vesi-
cles are also stable (stability > 60 days), which can be used
as drug carriers in pharmaceutical industry. The vesicles are
also stable with time in the presence of additives like so-
dium chloride, cholesterol etc.
In order to investigate drug entrapment ability and pH-
induced release by the vesicle structures, calcein was usedFig. 2. Negatively strained (1% uranyl acetate) transmission electron
micrograph of the SLS-CTAOH (1 mM, X1 ¼ 0.2) mixture.
S. Ghosh et al. / C. R. Chimie 19 (2016) 951e954 953as a model drug. The pH-sensitive dye calcein was chosen
as its four carboxylic groups get deprotonated at high pH.
The tetra anion due to its low permeability remains
entrapped inside the vesicles during these measurements.
Consequently, fluorescence intensity of calcein decreases
with the decrease of solution pH and also with time.
Fluorescence spectra (Fig. 3(A)) of entrapped calcein
(lex ¼ 495 nm and lem ¼ 520 nm) were measured imme-
diately after dilution of the vesicle phase in a cationic-rich
(X1 ¼ 0.20) mixture of the SLS-CTAOH system in buffered
solution of desired pH. Due to the decrease in solution pH
vesicles are disrupt and calcein is released as shown by the
quenching of fluorescence intensity (Fig. 3(A)). The
entrapment and slow release of water-soluble drugs are
thus demonstrated by the vesicles in the SLS-CTAOH
mixture.
The long term stability of the dye-encapsulated vesicles
was also considered. As time progresses, it is expected that
the encapsulated dye would leak through the vesicle
bilayer into the bulk solution. Due to self-quenching the
fluorescence intensity of vesicle-entrapped dyes is very
weak. However, the release of calcein from the vesicle core
will enhance fluorescence intensity. The time-dependent
increase of the fluorescence emission intensity of calcein
was taken as a direct measure of the calcein efflux rates
from vesicles. This is illustrated in Fig. 3(B), which shows
the time trace obtained over the course of two months.
Finally, the disruption was observed to be completed with
the addition of Triton X-100, a nonionic detergent that
disrupts both lipid and surfactant vesicles. Fig. 3(B) shows
the % leakage of entrapped calcein from the vesicles formed
by the cationic-rich SLS-CTAOH mixture (X1 ¼ 0.20). It is
seen that only 30e35% of the entrapped dye molecules
were released during the period of observation. The rela-
tively slow release suggests that the dye or drug entrap-
ment and its subsequent release are not influenced largely
with time and can be easily applied to the pharmaceutical
industry.
In order to investigate the bioavailability of vesicles as
vehicles in drug delivery applications, hemolytic activityFig. 3. (A) Fluorescence spectra of vesicle-entrapped calcein in 10 mM SLS-CTAOH (
extent of calcein released from SLS-CTAOH (X1 ¼ 0.20) mixed vesicles at pH 7.4 aswas determined. The hemolytic activity is assessed by
determining the hemoglobin release during the process of
destruction of hematid. The results described later suggest
that the vesicles can be efficiently used as a vehicle as they
are found to be hemocompatible and do not show any
hemolysis. However, before using in vivo, it is very much
desirable to explore the compatibility of the surfactants
with blood components. To evaluate this, hemolytic assays
were performed for the three mixed surfactant systems
SOS-CTAOH, SDS-CTAOH, SLS-CTAOH, and STS-CTAOH in
two different compositions (X1 ¼ 0.2 and 0.8) at pH 7.4
(HEPES buffer, 20 mM). Freshly isolated human red blood
cell (RBC) suspension (5% v/v) was added to HEPES-
buffered saline, 1% Triton X-100 and surfactants with a
final concentration of 5 mM, and incubated for 60 min at
37 C. The hemolytic activity (in %) of the different surfac-
tant systems along with the þVe and eVe control are
presented in Fig. 4. As seen, clearly none of the samples
exhibits any hemolysis. It is known that any samples with
less than 5% hemolysis ratio are nontoxic [14]. In the pre-
sent investigation, it is observed that the surfactants SOS-
CTAOH, SDS-CTAOH, SLS-CTAOH, and STS-CTAOH
(X1 ¼ 0.20) at a concentration of 5 mM exhibit 2.8 ± 0.11,
2.2 ± 0.05, 1.9 ± 0.11, and 1.5 ± 0.12% hemolysis, respec-
tively. Therefore, surfactant mixtures containing different
amino acid based anionic surfactants did not exhibit any
significant lysis to the RBC membrane and hemolytic ac-
tivity of the surfactants was hardly dependent on the chain
length of the anionic surfactant. When the surfactant
composition was changed to X1¼0.8 the hemolysis was
found to decrease to 2.3 ± 0.08, 1.9 ± 0.18, 1.6 ± 0.13, and
1.3 ± 0.11%, respectively. However, the values (<5%) still
remain within the limit of toxicity. As seen, clearly none of
the samples exhibits any hemolysis in comparison to eVe
control, that is, the surfactant mixtures containing amino
acid-based anionic surfactants did not exhibit any signifi-
cant lysis to the RBC membrane. Thus it can be concluded
that the mixed surfactant vesicles can be efficiently used as
a vehicle as they are found to be hemocompatible and do
not give any hemolysis.X1 ¼ 0.20) containing 10 mol % cholesterol at different pH values; (B) plot of
a function of time.
Fig. 4. The plot of % Hemolysis of the 5 mM mixed surfactants at X1 ¼ 0.20
and 0.80 at physiological pH (7.4). The bars indicate the means ± SD (n ¼ 3).
S. Ghosh et al. / C. R. Chimie 19 (2016) 951e954954The biocompatibility of the surfactant mixtures was
further evaluated by measuring the cell viability against
human cells. The cytotoxicity of the surfactants (5 mM) in
two different compositions (X1 ¼ 0.2 and 0.8) at physio-
logical pH (7.4) was evaluated in order to identify their
potential use in drug delivery applications. Cell viability
was determined by measuring the viability of 3T3 cells
using the MTT assay, based on the uptake and reduction of
the soluble yellow MTT tetrazolium salt by mitrochondrial
dehydrogenases to a blue insoluble MTT formazan product.
The cell viability is shown in Fig. 5. No significant toxicity
can be observed even at different surfactant compositions
and more than 85% cells are still found viable after 48 h of
treatment. To quantify the hemolytic effect of each treat-
ment, the percent cell lysis relative to the untreated cell (%
control) was determined by measuring the absorbance
(540 nm) of the supernatant solution. Hence, the biocom-
patibility of the presently investigated surfactant systems
was judged to be excellent. They are compatible to blood
component RBCs and do not affect the cell growth andFig. 5. MTT assay based fibroblast cell line 3T3 cell viability (in %) as a
function of surfactant composition (X1 ¼ 0.20 and 0.80) at physiological pH
(7.4). The bars indicate the means ± SD (n ¼ 3).therefore can be used in pharmaceutical applications like
drug carriers.
4. Conclusions
These unilamellar catanionic vesicles formed by the
binary mixtures of amino acid-based surfactants with
CTAOH (or DTAOH) have hydrodynamic diameter in the
range of 30e200 nm and drug entrapment ability of the
vesicles suggest their stability at room temperature for a
longer period of time. However, with the decrease in pH
(pH 5) the mixed surfactant vesicles are transformed into
small mixed micelles. This means that the vesicles are
sensitive to pH change of the environment. These pH-
sensitive vesicles are potentially interesting as drug de-
livery systems inwhich one aims to trigger the drug release
from the vesicles as the pH is decreased from a neutral or
slightly alkaline pH to an acidic pH. Bioavailability of the
vesicles was determined to consider the formulation of a
new encapsulating system as a vehicle in drug delivery
applications. The biocompatibility studies, namely cyto-
toxicity and hemolytic potential, of the spontaneously
formed pH-sensitive mixed surfactant vesicles have been
evaluated. The mixed surfactant vesicles were found to be
hemocompatible and did not show any hemolysis (<5%)
against red blood cells. MTT assay also did not suggest any
significant toxicity for any of the surfactant mixtures and
~90% cells were found viable even after 48 h of treatment.
The catanionic vesicles, because of their small size, stability,
and nontoxicity to the blood component RBCs as well as
mammalian cells can find applications in pH-triggered drug
delivery. In fact, entrapment of water-soluble dyes into the
vesicle core and its subsequent release triggered by pH
change has been demonstrated in this work.
Acknowledgments
Authors gratefully acknowledge Central Research Facil-
ity, IIT Kharagpur, India for instrumental assistance.
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Can a catanionic surfactant mixture act as a drug delivery vehicle?

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