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R. Kant, G. Bhanjana, N. Dilbaghi, S. Guru, S. Bhushan and S. Jaglan, RSC Adv., 2016, DOI:
10.1039/C6RA09677H.
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Page 1 of 35
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DOI: 10.1039/C6RA09677H
Gurpreet Kaura*, Sandeep Kumarb, Ravi Kantb, Gaurav Bhanjanab, Neeraj Dilbaghib, Santosh
Kumar Guruc, Shashi Bhushanc, Sundeep Jagland
Department of Bio and Nano Technology, Guru Jambheshwar University of Science &
Quality Control & Quality Assurance Division, CSIR-Indian Institute of Integrative Medicine,
______________________________________________________________________
Corresponding author: (Gurpreet Kaur)
Tel.: +91-9872800434, +91-2534431
E-mail address: gurpreet14@pu.ac.in
Anticancer Material
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Abstract
In this work a silver based double chained metallosurfactant was synthesized and characterized
using Fourier transform infrared (FTIR), elemental analyses, X-ray diffraction (XRD),
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Introduction
Metal ions have a very crucial role in a number of biochemical/biophysical processes [1].
and intracellular availability. Apart from this, they are potential candidates as anticancer agents.
The driving force towards the applicability of metal-containing compounds as anticancer drugs is
revolutionized with cisplatin [2]. However, cisplatin suffered from various disadvantages for
example toxicity towards healthy cells, its chemical instability (subject to facile hydrolysis under
low Cl- and/or low pH condition), and its poor water solubility [3,4]. Various attempts have been
made to increase the therapeutic efficacy of cisplatin and other cancer drugs by solubilizing them
in hydrophilic polymers or by embedding in liposomes or functionalized nanoparticles [5-9].
To comprehend, the dual functional moieties have raised the interest of researchers, one
of such kind are metallosurfactants (metal based surfactants). Metallosurfactants offer
advantages of metals which imparts properties such as acid-base, redox and magnetic and also
give amphiphilic character to the molecule. The metal based colloidal assemblies itself possesses
therapeutic activity and at the same time can act as a carrier if required. Although the property of
micellization of conventional anionic surfactants in the presence of divalent metal ions in
aqueous solution was known for quite some time but a metallosurfactant containing a surfactant
in a solid state was reported by Fallis et al [10] for the first time in 1998. The physicochemical
properties, aggregation behavior and biological activity of metallosurfactant depend on the type
of metal present and structure of metallosurfactant [11], however, they sometimes suffer from
poor water solubility [12]. Over the years, metallosurfactants have been synthesized and have
found applications in diverse fields such as catalysis, anti-cancer activity, the formation of
lamellar superstructures, DNA templates, and templates for metallic mesostructures, etc [11-14].
3
Metals show distinctiveness in redox activity, variable coordination modes, variable reactivity
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DOI: 10.1039/C6RA09677H
For
example,
the
surfactantcobalt
(III)
complex
(metallosurfactant)
cis-
[Co(trien)(C14H29NH2)Cl](ClO4)2 was evaluated for its cytotoxicity on the MCF-7 breast cancer
properties and causes damage to DNA. On the other hand, Adawy and Khowdiary reported
antimicrobial activities of various cationic metallosurfactants [16]. Likewise, other
multifunctional materials have also been reported for e.g. a multifunctional block copolymer
assembly was fabricated by Osada et al [17] that along with fluorescent imaging of the tumor,
possesses an appreciable antitumor activity as well. Zhang et al [18] have prepared antibacterial
vesicles from a block copolymer that act as a carrier for an anticancer drug.
The aim of the present work is to optimize a dual functional assembly of metallosurfactant
that can self-aggregate and have biological activity. Therefore, in this work we had prepared a
silver based metallosurfactant that can self-assemble and can act as a carrier (if required) and
also have therapeutic activity (i.e. anticancer). But most importantly it possesses anti-microbial
activity so that the enhanced efficacy can be achieved by fighting with the microbial infections
during or after chemotherapy, which is a commonly observed. For enhanced activity the choice
of metal was crucial and for the purpose silver was chosen because of well known biocidal
effects of silver ions, its compounds, colloids, and nanoparticles [19-20]. Furthermore, it was
extremely important to understand the mechanism of action of silver as metallosurfactant against
various microbial strains.
Long chain alkylamines are widely used as the templates to synthesize the mesoporous
materials in single or binary solutions because they could self-assemble into different structures
[21-23]. A cetyltrimethylammonium silver bromide complex was reported by Liu et al, where
the reaction of silver nitrate with cetyltrimethylammonium bromide (CTAB) in aqueous solution
4
cells [15]. It was found that the complex possesses antiproliferative and apoptosis-induction
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DOI: 10.1039/C6RA09677H
at room temperature was performed, by controlling the concentration and the molar ratio of
CTAB to silver nitrate [24].
silver based water soluble metallosurfactant was synthesized. To achieve this, a straightforward,
high yielding protocol was developed using ligand insertion method. The surfactant was
characterized using elemental analyzes, FTIR, NMR, TGA, and XRD. Further, the selfaggregation property of the metallosurfactant was determined by using a combined approach,
where self aggregation was studied using surface tension, conductivity and morphology with
TEM and AFM. The bioactivity profile of this complex was investigated on cancer (pancreatic,
prostate and leukemia) and normal cell lines using MTT assay. The antimicrobial properties were
also evaluated on different bacteria and fungi samples using well diffusion assay. To account for
its anticancer and antimicrobial properties, its interaction with calf thymus DNA (CT-DNA) was
also evaluated spectrophotometrically. The results obtained have shown that the bioactivity was
enhanced with the incorporation of the silver counter ion. Moreover, the silver based
metallosurfactants are found to be less toxic because four times high IC50 value in normal cells
was obtained in comparison to cancer cells.
Material and methods:
AgNO3 (99.0%), CTAB (99.0%), calf thymus DNA (purity98%), Ethidium bromide (EB)
(purity95%), tetramethylsilane (99.4%) EDTA, DMSO, DCM, tetrazolium dye MTT 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DMEM (Dulbeccos Modified Eagles
Medium), EMEM media containing 5% foetal bovine serum (FBS), NAD(P)H, MEM Medium
containing 10% FCS trypsin, RPMI-1640, kanamycin, streptomycin and CDCl3 (99.2%) were
purchased from Sigma Aldrich. Absolute ethanol (99.9%) was purchased from Changshu Yuang.
5
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nutrient agar and potato dextrose agar were purchased from HiMedia. The fungal
strains Curvularia lunata (ITCC 6257) Helminthosporium oryzae (ITCC 5559), Aspergillus
used for the present study were procured from the Indian Type Culture Collection (ITCC), Indian
Agriculture Research Institute (IARI), New Delhi, India. Escherichia coli (MTCC 40) and
Staphylococcus aureus (MTCC 2901) were obtained from IMTECH Chandigarh. Human
leukemia HL-60, pancreatic MIA-Pa-Ca-2, prostate cancer PC-3 cells and normal cells (fR2
human breast epithelial cells) were obtained from ECACC, UK.
Precautions were taken while handling Ethidium bromide. The safety data sheets were also
consulted for DMSO and DCM.
Procedure for the formation of metallocomplex:
The Ag metallocomplex (CTA-AgB) was prepared by refluxing the metal salt (0.5 mmol) and
ligand i.e. CTAB (1 mmol) in 1:2 ratio. The AgNO3 was dissolved in absolute ethanol (5 ml)
with subsequent addition of ligand i.e. CTAB. The mixture was stirred for 2 h at 150 rpm. The
product is obtained after evaporating the solvent. Recrystallization of the final powdered product
was carried out in methanol.
Physical measurements
Eager Xperience CHN was used for elemental (CHN) analyses. The experiment was performed
under inert nitrogen medium. FTIR spectra were recorded on Perkin Elmer (RX1) spectrometer
in the far-IR range of 200 to 600 nm and IR range from 600 to 3500 nm. For each spectrum, 24
scans were recorded with a spectral resolution of 4 cm-1 on KBr pellets. Thermogravimetric
analyses (TGA) were carried out using SDT-Q-600 (TA instruments). A sample mass
(approximately 5-10 mg) was transferred to aluminum crucibles and analyzed from room
6
fumigates (ITCC 1628), Aspergillus niger (ITCC 302), Cladosporium herbarum (ITCC 3137),
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temperature to 1000C at a heating rate of 10C min-1 under the nitrogen atmosphere. Differential
Scanning Calorimetry (DSC) measurements were done using TA (Q20) in aluminum pans with
cooled in liquid nitrogen up to 100C. After equilibration, the samples were heated at 10C
min1 to reach the ambient temperatures. X-Ray Diffraction (XRD) spectra were recorded on
Panalytical XPert Pro X-ray diffractometer equipped with Cu-k radiation (1.5406 ) operating
at 40 kV, with a scanning speed of 8/min to examine the crystalline phase of the sample. For 1H
NMR studies, JEOL FT-NMR, AL 300 was employed with 300 MHz radio frequency and 7.05tesla magnetic field strength. Tetramethylsilane was used as internal standard and D2O was used
as a solvent.
Self aggregation
Critical micellization concentrations (cmc) were determined through conductivity measurements
performed on Pico Lab India Digital conductivity meter using doubly distilled water
(conductivity less than 5Scm-1). These studies were carried out for a temperature range of 25-40
C. The measuring cell and sample were dip into jacket whose temperature (0.01C) was
maintained through the thermostat. Surface tension of prepared metal complex was measured at
25C with Du Nouy tensiometer (Kruss type 8451) using double distilled water. The surface
tension of the used double distilled water was 71 mN/m at 25C. TEM micrographs of
metallomicelles were obtained using Hitachi (H 7500) instrument at an accelerating voltage of
90 kV. Freshly prepared samples were gently placed over 200-mesh formwar copper grid
uniformly coated with carbon film. AFM studies were performed using Bruker Nanoscope Vmultimode 8 with an operating frequency of 312.1309 kHz and tapping mode was used for
image scanning. Multiply filtered metalomicelles were gently sonicated for 2 h prior to sample
7
the blank pan as a reference (maintained at the same temperature). The samples were rapidly
RSC Advances
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preparation for AFM study. An aqueous solution of metallomicelles was gently poured on
ultrapure silicon wafer 1cm 1cm and was dried overnight in a vacuum desiccator to obtain a
Perkin Elmer spectrophotometer with matched pair of 1cm quartz cell fitted in thermostatic cell
holder. Absorption spectra were carried out between 200 and 800 nm. Fluorimetric studies of
CT-DNA were carried out in the presence of a variable concentration of metallosurfactant on
Hitachi F-7000 Photo luminescence spectrophotometer. The excitation wavelength was set at
450 nm, with a slit width of 10 nm and range of observation was selected between 500-700 nm.
Antimicrobial studies
In vitro antimicrobial tests were carried out using well diffusion assay against different
microorganisms [25,26]. Bacteria were cultured in nutrient agar broth and fungus was cultured in
potato dextrose agar broth media incubated at 37 and 25 C respectively for 24 h. 60 L (0.5
mg/mL, aqueous) of test samples were added to 6 mm well bored on agar plates after
homogeneous pouring of freshly cultured microbial stains. After overnight incubation, diameters
of inhibition zones were measured in nearest of millimeters and the average value of triplicate
measurements was reported. The diameter of the well was excluded from inhibition zone
diameter. Effect of concentration on microbial growth was also studied by using multiple
dilutions of the test samples. Mechanism of action of synthesized metallosurfactant against
various microbial strains was studied using TEM microscopy by checking interaction of
metallosurfactant and microbe at regular time intervals.
Cytotoxic studies
Cell culture and treatment
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The cells were grown in RPMI-1640, DMEM and MEM medium containing 10% FCS, 100
g/mL kanamycin and streptomycin. Cells were grown in a CO2 incubator (Thermocon Electron
monolayer cultures were trypsinised with trypsin (0.1% w/v)/EDTA (1 mM) solution. Soon after
cells were ready to detach, the trypsin/EDTA solution was removed. Cells were dispersed gently
by pipetting in complete growth medium, centrifuged at 200xg, for 5 min. Cells were dispersed
in a complete medium in culture flasks and incubated in the CO2 incubator. Cells grown in semiconfluent stage (approx. 70% confluent) were treated with CTA-AgB dissolved in DMSO while
the untreated control cultures received only the vehicle (DMSO, < 0.2%).
MTT Cell Proliferation Assay:
This assay is a quantitative colorimetric method for the determination of cell survival and
proliferation. This assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent
cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable
cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a
purple color. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable
cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents
and toxic materials. The assessed parameter is the metabolic activity of viable cells.
Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to a dark blue waterinsoluble formazan, which can be, after solubilization with DMSO, directly quantified. The
absorbance of the formazan directly correlates to the number of viable cells. The cells were
seeded at a density of 15,000(HL-60), 6000(PC-3) and 5000(MIA-Pa-Ca-2) plated in 96-well
plates. Cultures were incubated with 1, 3, 5, 7 and 10 M concentrations of test materials and
9
Corporation, USA) at 37C with 5% CO2 gas environment and 95% humidity. Cells grown in
RSC Advances
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incubated for 48 h. After 44 h, 20 L of MTT dye was added at a concentration of 2.5 mg/mL for
4 h. The supernatant was aspirated and MTT-formazon crystals dissolved in 150 L of DMSO;
ELISA reader (Thermo Laboratories, U.S.A). Cell growth was calculated by comparing the
absorbance of treated versus untreated cells [27].
Results and discussion
To prepare CTA-AgB complex in 1:2 ratio of silver nitrate to CTAB, which readily
resulted into formation of white colored CTA-AgB complex due to enhanced surface adsorption
without formation of silver bromide nanoparticles (yellow in colour) [24]. Secondly, the
formation of nanoparticles occurs at a lower molar ratio of CTAB (for e.g. 1:0.5), it has been
reported that for ratio more than 1:1.5 only complexation takes place [24]. The structure of the
complex is given in Figure 1 (a) and characterized with the help of various analytical techniques.
The elemental analysis (Found: C=51.20, H=9.49, N=4.86) is in good agreement with the
calculated value (Cald: C=50.73, H=9.35, N=4.67).
The complexation of ligand to metal salt was confirmed by comparing the FTIR spectra
of the pure ligand with metallocomplex. Figure 1(b) depicts the spectra of pure ligand and the
prepared metallocomplex. Table S1 gives the FTIR data of pure ligand and metallocomplex.
CTAB has 16 carbon chain, therefore, the two intense bands at around 2915 and 2848 cm-l are
assigned to asymmetric and symmetric stretching vibrations of C-CH2 from the methylene
chains, respectively. After complexation with a metal salt, no change was observed in the
hydrophobic chains of ligand. For CTAB, the bands at 1484 and 1473 cm-1 correspond to
asymmetric and symmetric CH scissoring vibrations of CH3-N+. A shift from 1473 to 1464 cm-1
was observed [28]. Bands at 1382 and 1258 cm-1 represent the N-CH3 symmetric stretching
10
optical density was measured at a wavelength of 540 nm (reference wavelength, 620 nm) on an
Page 11 of 35
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vibrations. The complexation of AgNO3 and CTAB is well adjudged from the shifts in peaks
from two bands 1407 and 1382 cm-1 to a single peak at 1332 cm-1, whereas 1258 cm-1 disappears
the spectra are clearly seen from that of the earlier reported silver capped nanoparticles. The
terminal Ag-Br peaks are observed in the far-IR region at 170, 215 and 49 cm-1 [29]. In the
present complex, the bridge Br stretching vibrations at 231 and 112 cm-1 further confirms the
complexation (Figure 1b) [30]. The metallosurfactant was further characterized by XRD and
DSC (Figure 1 (c) and (d)). Similar XRD pattern and DSC curves have been obtained as reported
[22,24,31], CTAB exhibited a sharp endothermic peak at 108 C, whereas, the peak for
metallosurfactant appeared at 98C which attributed to the melting of the ordered regions of
hydrocarbon chains in pure CTAB and CTA-AgB. In NMR spectra of CTAB (Figure S1 (a),
chemical shift at 3.082 and 3.338 was assigned to -CH3 and '-CH2, respectively. For '-CH2 CH2, and '- CH2, was obtained at 1.671, 1.270, and 1.188, respectively. The terminal methyl,
-CH3, gave chemical shift at 0.765. The comparison of
metallosurfactant (Figure S1 (b)) was made and values (Table S2) clearly depict the change
in the position of protons around the head group i.e. -CH3 and '-CH2, and the change of
counter ions are clearly evident [21].
Further, TGA characterization has been carried out and TG curves of pure CTAB and CTA-AgB
are given in Figure 2 (a). The TGA curve of pure CTAB initially shows loss of water around 100
C. CTAB is known to decompose within 220350 C [23]. The first step of decomposition was
observed at 247.15 C for CTAB and DTA peak at 275.3 C for Ag complex and another event
at 426 C observed was assigned to the melting of silver bromide crystals. The rate of
11
completely. No change was observed at 1151, 1037, 959 cm-1 on complexation. Distinctions in
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decomposition of CTAB and its Ag complex has been estimated using the non-isothermal
thermogravimetry (TG) method. The values of activation energy, Ea, and reaction order, n of the
DTG
T (C)
247.15
CTA-AgB
275.3
CR
E/kJmol-1
(R)
11.81
(0.999)
28.05
(0.999)
MKN
E/kJmol-1
(R)
11.96
(0.999)
28.21
(0.999)
WYHC
E/kJmol-1
(R)
12.01
(0.999)
28.26
(0.999)
12
VK
E/kJmol-1
(R)
13.85
(0.998)
32.59
(0.999)
HM
E/kJmol-1
(R)
15.88
(0.999)
32.58
(0.999)
order
Zero order
Zero order
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(a)
'
'
'
'
'
'
'
CTAB
'
'
'
'
'
'
'
Bromide
Silver
(b)
%T
CTA-AgB
CTAB
100
200
300
400
600
500
700
800
900
1000 1200 1400 1600 1800 2000 2500 3000 3500 4000
-1
wavenumber (nm )
1600
(c)
(d)
1400
-1
Heat Flow (W/g)
1200
1000
800
600
CTAB-Ag
CTAB
-2
-3
-4
400
-5
200
0
-6
10
12
14
16
18
20
50
100
150
200
Temperature ( C)
Figure 1: (a) Structural representation of CTAB and CTA-AgB (b) FTIR (50-4000 cm-1) of
CTAB and CTA-AgB (c) XRD spectra of CTA-AgB (d) DSC of CTAB and CTA-AgB
13
CTAB-AgB
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Self aggregation
Self aggregation of molecules is outplay of various interactions i.e. hydrophobic interactions
with oppositely charged counter ion), hydrogen bonding (among solvent molecules) and
sometimes metal ligand interaction (as is the present case) [33,34]. Micellization is also
influenced by other parameters such as temperature, dielectric constant etc [35,36]. To
understand micellization in the present work, where a metal ion is present as a counter ion having
2:1 stoichiometry of hydrophobic chains and counter ions, (which was altered from usual 1:1 for
CTAB) conductivity and surface tension measurements were performed. Figure 2 (b, c, d) gives
the variation of specific conductance, molar conductance and surface tension with a concentration
of metallosurfactant. Specific conductance increases with increase in the temperature. The cmc
determined by surface tension measurement was in good agreement with the one obtained from
conductivity studies. In previously reported work, micellization behavior of metallosurfactants in
both aqueous and nonaqueous medium showed a decrease in cmc in comparison to the parent
conventional surfactant because of crowding at the interface [32,37,38].
Similar behavior was expected for this silver metallosurfactant with AgBr2 as a counter
ion because metallic counter ion is bigger in size and is expected to undergo weak hydration,
therefore, lower cmc was anticipated but the results obtained were contrary. The cmc of CTAB
at 25 C is reported between 0.84 and 0.97 mM [39,40] and CTA-AgB complex displayed
almost equivalent cmc values as compared to CTAB except for 40 C (Table S3). Jakubowska
[41] has recently explained counter ion binding with different anionic headgroups, where the
types of counter ion (kosmotropic and chaotropic ions) have shown different types of binding to
monomers and dimers. Similarly, in this case, Ag being a kosmotropic ion shows strong ion
14
(among surfactant tails), electrostatic interactions (repulsive between head groups and attractive
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hydration. The kosmotropic ions are usually small, have relatively low polarizabilities, therefore,
they lose their water of hydration with great difficulty. These types of ions are strongly screened
in this case), than the expected decrease. The increase observed was slight may be because
precursor CTAB itself has a large bromide ion, therefore, a significant difference in the cmc
values was not observed because of the simialr hydration of both the ions.
100
(b)
140
(a)
25
30
35
45
120
-1
(Scm )
80
weight loss (%)
160
Ag-CTAB
CTAB
60
40
100
80
C
C
C
0
C
0
0
60
40
20
20
0
100
200
300
400
o
Temp ( C)
500
0.0
600
100
90
1.0
1.5
Conc. (m M )
2.0
2.5
55
(c)
(d)
50
80
-1
(mN m )
70
60
25
30
35
40
50
40
C
C
0
C
0
C
0
45
40
35
30
20
0.4
0.5
0.6
0.8
1.0
1.2
[Conc.(mM )]
1.4
-1.2
1.6
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
log conc
1/2
Figure 2. (a) TGA of CTAB and CTA-AgB (b) variation of specific conductance (c) molar
conductance and (d) surface tension with concentration of CTA-AgB
15
and is strongly determined by the ion hydration, therefore, results in higher cmc (although slight
d/dc
due to strong ion hydration, the binding of these ions to interfaces by electrostatic forces is weak
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variation was observed for interaction parameter (), and H om . The surfactant ionization
increases with increase in the temperature; this illustrates the decrease in the counter ion binding
due to thermally aggravated process. The variations for H om and Som were quite sensitive to
temperature. The value of H om decreases with increase in the temperature and remain negative
throughout. However, Som decreased with temperature and remained positive. G om is the sum
of the enthalpic ( H om ) and entropic (-T Som ) contributions, with an increase in the temperature,
the enthalpic contribution to the free energy increases, whereas the entropic contribution
decreases (Figure S5), however, the process is overall entropy driven. The Gibbs surface excess
tot
at cmc ( max
) was calculated from the linear part of the vs. log [Ct] plots, as shown in Figure 2
1
d
lim [C ] cmc
2.303nRT
d log[C]
(1)
where n, R and T are the number of ionic species per surfactant monomer in solution, the
universal gas constant, and absolute temperature, respectively. In this equation, the concentration
was used in place of activity since the solutions in use were fairly dilute. The area of exclusion
per monomer (Amin) at the saturated air/solution interface was calculated by using eq. (2)
A min =
10 20
tot
Nmax
(2)
The surface excess along with minimum area occupied by a head group decides the extent of
packing
at
the
air/water
interface.
In
16
the
case
of
Ag
metal
surfactant,
by phase separation model using equations S1-S3 given in supporting information. Linear
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tot
max
= CTA + Br + Ag + + NO with a value of 0.75 mols m
3
-2
tot
comparison to CTAB ( max
=1.68 mols m-2; A min = 99.2 2) was found to be low and this is
The presence of metal ion at the interface is expected to enhance the steric crowding at the
interface. Wherein, micellization in the present case is mainly being controlled by the counter ion
hydration.
The micellization of CTA-AgB was viewed using TEM and AFM. Figure 3 shows the
aggregates of metallosurfactant prepared at twice concentration of cmc, with an average size 6
nm (Figure 3a) and AFM clearly depicted the spherical shape of these aggregates (Figure 3b).
After the characterization of CTA-AgB micelles, its interaction with CT-DNA was estimated
using absorption and emission spectroscopy. Fluorescence quenching experiments give us
information regarding localization and the mode of binding with DNA. 3, 8diamino-5-ethyl-6phenyl-phenanthridinium Bromide (EB) a cationic dye is widely used in spectrofluorimetric
studies as nondestructive fluorescent intercalator displacement (FID) assay due to fluorescence
enhancement it displays upon binding with DNA. Fluorescence enhancement is due to
intercalation of the dye into double helix conformation of nucleic acid [42]. The continuous
decrease in fluorescence intensity with the addition of surfactant (0-45 M) is indicative of
replacement of intercalated EB (4 M) molecules from DNA (75 M) (Figure 3c). It was found
that intercalating agents, as well as groove binders, cause a reduction in emission intensity,
however, moderately in the present case. Binding affinity was evaluated by applying Stern
Volmer equation and a high Ksv (binding constant) value (3.20*104 LM-1) show that
metallosurfactant bound strongly to the DNA (Figure 3d). In UV-visible absorption
17
ascribed to the competitive adsorption between the cationic head group and metallic counter ion.
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carried out with increasing concentrations, which did not show any shift (Figure S6), thereby
confirming hyperchromism effect is due to complexation of DNA with metallosurfactant. This
might be because of the electrostatic interactions between metallosurfactant and CT-DNA, which
embodies changes in the conformation of CT-DNA. Such increase in absorption intensity is
generally due to the exposure of purine and pyrimidine DNA-bases on the binding.
Quantitatively binding affinity, as well as intrinsic binding constant Kb, is calculated using the
equation (3)(3)
[ DNA ] /( a f ) = [ DNA ] /( f ) + 1 / K b ( f )
where [DNA] is the concentration of DNA ; a, f and 0 are the apparent, free and fully bound
Ag complex extinction coefficients. A plot of [DNA]/(a f) versus [DNA] gives Kb as the
ratio of the slope to intercept. High Kb values indicate a strong electrostatic interaction between
metallosurfactant and CT-DNA (Figure 3f).
(a)
(b)
18
formation of complex with CT-DNA (Figure 3e). The UV-visible spectra of DNA alone were
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0 M
(d)
0.25
(e)
(f)
15
0.20
Kb = 3.456 * 10 LM
-1
0.15
1
0.10
0.05
0.00
200
225
250
275
300
325
wavelength (nm)
Figure 3. (a) TEM and (b) AFM of metallomicelles; (c) and (d) ct-DNA interaction with
metalomicelles using fluorescence spectroscopy; (d) and (e) using UV-visible spectroscopy
Further, we explored the utility of the silver metallocomplex in killing microbes and cancer cells
using in vitro analyses by employing well diffusion and MTT assay, respectively.
Cytotoxicity activity: Nanoscale complexes currently being developed consist of two main
components: the nanoparticle itself, which is used as the carrier agent, and the chemotherapeutic
drug. Studies using paclitaxel have shown that when compared with the conventional
formulation, the nanoparticle formulation of the drug increases both its cytotoxicity profile in
cell culture and its therapeutic efficiency in a living animal model. This has been attributed to the
nanoparticle formulation having greater bioavailability and a longer sustainable therapeutic time,
which allows the drug concentration to remain above the minimum effective value for an
19
45M
Absorbance
(c)
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extended period of time [43]. In the present work we are proposing a dual functional colloidal
assembly which itself possess anti-proliferative activity. CTA-AgB complex was tested for anti-
120
100
80
60
40
20
0
0
2
3
Concentration (M)
Figure 4: Percentage growth inhibition of different cancer lines using CTA-AgB as analyzed by
MTT assay
20
prostate PC-3 and human leukemia HL-60 cancer cell lines (Figure 4), in the concentration range
proliferative activity in three different cancer cell lines including pancreatic MIA-Pa-Ca-2,
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Ag Nps prepared
from extract of
Alternanthera
sessilis
AgNPs
Ag Nps prepared
from extract of
Eucalyptus
chapmaniana
Nano Ag sol
Ag Nps
46
IC50: 2 mmol/L
47
Human promyelocytic
leukemia cells (HL-60)
; MTT assay
Human promyelocytic
leukemia cells (HL-60)
; MTT assay
Viability cells
diminished to 60 % at
dose of 25 mg/l
Viability cells
diminished to 40 % at
dose of 0.5 mM AgNPs
48
Ref
44
45
49
Antimicrobial activity:
To access the antimicrobial activity of the CTA-AgB complex, agar well diffusion method was
employed. Two bacterial (E. coli as Gram-negative and S. aureus as Gram positive) and five
fungal strains (A. niger, A. fumigates, C. herbarium, C. lunata and H. oryzae) were considered
for antimicrobial studies. Bacterial strains were grown in nutrient media at a temperature of 37C
and for fungus, potato dextrose media was used at a temperature of 25 C. Metallosurfactant in a
concentration of 0.5 mg/ mL were used. Zones of inhibition obtained for this concentration are
presented in Table S4, where the same dose of 0.5 mg/mL of silver metallosurfactant gave a
comparable average zone of inhibition. Effect of metallosurfactant concentration on microbial
21
Nanoparticles
Ag (+) NPs,
Ag (-)NPs
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growth was also studied by counting visible colonies and obtained results are shown in Table S4
for five different concentrations. At some dilutions the growth of colonies was so vigorous that
Moreover, it is also clear from the Table S4 that the primary used concentration (0.5 mg/mL) is
also the MIC (minimum inhibitory concentration) for E. coli species. As far as dilutions of fungi
are concerned, lesser growth was observed for A. niger, A. fumigatus than the other fungi used in
the study.
no margins were observed, such growth was confluent, represented by C in the Table S4.
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cells have patches of dark appearance, these patches perhaps are cell organelles where
formulation got accumulated (left the cell in Figure. 5a and middle cell in Figure. 5a). Cells with
formulation to such an extent that whole of the cell appeared dark and no cell organelles could be
seen (left cell in Figure. 5b and 6b). So it can also be inferred that absorption of the formulation
is time dependent. Initially, absorption was inside some particular organelles but with the
passage of time this control on absorption was lost and formulations got accumulated in the
whole of the cell. Location of cells within bulk cell mass during treatment may account the same
variation in the localization of formulation. It is clear from the images (Figure. 5a, c and Figure.
6c) that the cells are oval in longitudinal section and circular in transverse sections. Membrane
perturbation was observed in almost all cells. E. coli is a peritrichous bacterium with flagella all
over its surface. These flagella were vanished by the action of metallosurfactant. This happened
in a very stepwise manner, as few cells are with small flagella (middle cell in Figure. 5c,d) and
many others are without flagella/small flagella. This damage to flagella was also time and
location dependent as described above. Further, E. coli with homogenous dark appearance lacked
flagella altogether (cells in extreme left in Figure. 5c). All the staphylococcus cells as expected
lacked flagellum (Figure. 6 a-f). Some cells depicted initial separation of plasma membrane from
cell wall while in many others there is complete separation (initial separation in upper cell and
complete separation in a lower cell in Figure. 6d). TEM analysis also discerned localized
separation. Gram-negative strains (Figure. 5e) were found to be more resistant to
metallosurfactant damage than Gram positive bacterial strains (Figure. 6). Metallosurfactant
initially came in contact with the cell wall and then subsequently penetrated inside.
23
homogenous dark appearance can also be seen from TEM images. These cells absorbed the
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in Figure. 7a), perhaps these areas were nuclei as A. niger is supposed to have many nuclei.
Cytoplasm had patches but it did not dissolve similar to bacteria whereas cell wall struggled till
its last to remain intact and maintained its shape (Aspergillus cells in Figure. 7a and 7b). In all
fungal cells except A. fumigates (Figure. 7c-e) there was no morphological loss to the cell wall.
A. fumigates cells were the only fungal cell (of all tested fungal cells) where cell wall gets
dissolved and cytoplasm was without a distinct boundary. In C. herbarium (Figure. 7 f,g),
dividing cells are showing shrinkage in DNA (transverse section of dividing cell in Figure. 7 g).
In A. Niger (Figure. 7 b), cell pits are formed, as this cell is appearing heterogeneous dark so it
can also be concluded that initially encounter in these cells allowed entry of metallosurfactant by
the formation of pits in the cell wall. There is also an accumulation of metallosurfactant at the
surface of cells and can be seen from the image (lower A. fumigatus cell in Figure. 7d). These
differences in bacterial and fungal cell walls perhaps are due to the different chemical
composition of two types of cells. Cytoplasm is less damaged in fungal cells (Figure. 7a-e) than
bacterial cells (right cell in Figure. 5c, Figure. 6 a, c, d). It is because cytoplasm of fungal cells is
more advanced than bacterial cells that maintained the integrity of its cytoplasm even in the
presence of metallosurfactant.
25
distinct, unlike bacteria. A large part of cytoplasm did not absorb the metallosurfactant (A. niger
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Figure 7. TEM images of (a,b) A. niger (c-e) A. fumigates (f,g) C. herbarium (h,i) C. lunata with
CTA-AgB metallosurfactant
Conclusion: A double chained silver based metallosurfactant was synthesized using CTAB as a
precursor. After confirming the complexation by various analytical methods, the rate of
decomposition of CTAB and its CTA-AgB was estimated using the TGA method. The activation
energy of decomposition of CTA-AgB was found to greater be than that of CTAB due to the
favorable softsoft interactions between bromide ions with silver ions. The metal based
surfactant was evaluated for its surface and self aggregation properties. It was found that cmc
was increased in the presence of silver ions and its formation is confirmed by TEM, AFM,
conductivity
and
surface
tension
measurements.
The
micellization
process
was
thermodynamically favorable with entropy being the driving force and is being influenced by Ag
hydration. The binding of metallosurfactant with CT-DNA was estimated using Uv-visible and
fluorescence spectroscopy, the results reveal a strong electrostatic interaction between the two.
CTA-AgB showed potential as an antimicrobial and anticancer agent. Estimation of anticancer
26
DOI: 10.1039/C6RA09677H
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years has proved to be one of the efficient antimicrobial agents in different forms, however, in
this its the dual functionality of silver based metallocomplex which of importance. TEM
analyzes were instrumental in understanding the mechanism of antimicrobial activity of the
prepared silver metal complex. Gram negative strains were found to be more resistant to damage
than Gram positive bacterial strains. Among fungi and bacteria, cytoplasm is less damaged in
fungal cells in comparison to bacterial cells. Scheme 1 gives the pictorial representation of the
formation of the silver metallosurfactant and its potential applications explored in this work.
27
Antimicrobial activity was evaluated using bacterial and fungal strains. Although silver over the
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Scheme 1: The pictorial representation of the formation of the silver metallosurfactant and its
potential applications.
GK is thankful to DST for Inspire Faculty award (IFA-12-CH-41) and DST PURSE grant II.
Authors from GJUS&T, Hisar thanks Nano Mission, DST, DBT and UGC for infrastructural and
research facilities and IIT Ropar for AFM analysis.
References
[1] Dudev, T. and Lim. C. Competition among metal ions for protein binding sites: determinants
of metal ion selectivity in proteins. Chem. Rev. 2014, 114, 538-56.
[2] Warad, I.; Eftaiha, A.F.; Al-Nuri, M.A.; Husein, A.I.; Assal, M.; Obaid, A.A.; Al-Zaqri, N.;
Hadda, T.B.; Hammouti, B. Metal ions as antitumor complexes-review. J. Mater.Environ. Sci.
2013, 4, 542-557.
[3] Florea, A.M. and Busselberg, D. Cisplatin as an anti-tumor drug: cellular mechanisms of
activity, drug resistance and induced side effects. Cancers 2011, 3, 1351-1371.
[4] Xu, C.; Wang, B.; Sun, S. Dumbbell-like Au-Fe3O4 Nanoparticles for target-specific ciplatin
delivery. J. Am. Chem. Soc. 2009, 131, 42164217.
[5] Cheng, K.; Peng, S.; Xu, C.; Sun, S. Porous hollow Fe3O4 nanoparticles for targeted delivery
and controlled release of cisplatin. J. Am. Chem. Soc. 2009, 131, 1063710644.
[6] Ramachandran, S.; Quist, A.P.; Kumar, S.; Lal, R. Cisplatin nanoliposomes for cancer
therapy: AFM and fluorescence imaging of cisplatin encapsulation, stability, cellular uptake, and
toxicity. Langmuir 2006, 22, 8156-8162.
28
Acknowledgement
Page 29 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA09677H
[7] Zhang, Y.; Wang, X.J., Guo, M.; Yan H.S. Cisplatin-loaded Polymer/Magnetite Composite
Nanoparticles as Multifunctional Therapeutic Nanomedicine. Chinese J. Polym. Sci. 2014, 32,
[8] Xu, P.; VanKirk, E. A.; Murdoch, W. J.; Zhan, Y.; Isaak, D. D.; Radosz, M.; Shen, Y.
Anticancer Efficacies of Cisplatin-Releasing pH-Responsive Nanoparticles. Biomacromolecules
2006, 7, 829-835.
[9] Oishi, M.; Hayashi, H.; Michihiro, I. D.; Nagasaki, Y. Endosomal release and intracellular
delivery of anticancer drugs using pH-sensitive PEGylated nanogels. J. Mater. Chem. 2007, 17,
3720-3725.
[10] Fallis, I.A.; Griffiths, P.C.; Griffiths, P.M.; Hibbs, D.E.; Hursthouse, M.B.; Winnington,
A.L. Solid state and solution behaviour of novel transition metal containing surfactants. Chem.
13291337.
RSC Advances
Page 30 of 35
View Article Online
DOI: 10.1039/C6RA09677H
[15] Riyasdeen, A.; Senthilkumar, R.; Periasamy, V. S.; Preethy, P.; Srinag, S.; Zeeshan, M.;
Krishnamurthy, H.; Arunachalam S.; Akbarsha M. A. Antiproliferative and apoptosis induction
49959.
[16] Adawy, A. I.; Khowdiary, M M. Structure and Biological Behaviors of Some Metallo
Cationic Surfactants. J. Surf. Deter., 2013, 16, 709-715.
[17] Osada, K.; Cabral, H.; Mochida, Y.; Lee, S.; Nagata, K.; Matsuura, T.; Yamamoto, M.;
Anraku, Y.; Kishimura, A.; Nishiyama, N.; Kataoka, K. Bioactive polymeric metallosomes selfassembled through block copolymermetal complexation. J. Am.Chem. Soc. 2012, 134, 1317213175.
[18]
E. coli a model for Gram-negative bacteria. J. Colloid Inter. Sci. 2004, 275, 177182.
[20] Liu, Z.; Wang, Y.; Zu, Y.; Fu, Y.; Li, N.; Guo, N.; Liu, R.; Zhang, Y. Synthesis of
polyethylenimine (PEI) functionalized silver nanoparticles by a hydrothermal method and their
antibacterial activity study. Mater. Sci. Engineer. C 2014, 42, 3137.
[21] Sui, Z.M.; Chen, X.; Wang, L.Y.; Xu, L.M.; Zhuang, W.C.; Chai, Y.C.; Yang C.J. Capping
effect of CTAB on positively charged Ag nanoparticles. Physica E 2006, 33, 308314.
[22] Ren, N.; Tang, Y.; Wang, Y.J.; Dong, A.G.; Yangy W.L. Fabrication of the sponge-like
layered silver(i)-alkylamine complexes and their in situ reduction. Chem. Lett. 2002, 372-373.
[23] Sui, Z.; Chen, X.; Wang, L.; Chai, Y. Yang, C., Zhao, J. An improved approach for
synthesis of positively charged silver nanoparticles. Chem. Lett. 2005, 34, 100-101.
30
studies of a metallosurfactant in human breast cancer cell MCF-7. RSC Adv. 2014, 4, 49953-
Page 31 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA09677H
[24] Liu, X.H.; Luo, X.H.; Luc, S.X.; Zhang, J.C.;, W.L. A novel cetyltrimethyl ammonium
silver bromide complex and silver bromide nanoparticles obtained by the surfactant counterion.
[25] Kaur, G.; Mehta, S.K.; Kumar, S.; Bhanjana, G.; Dilbaghi, N. Coencapsulation of
hydrophobic and hydrophilic anti-TB drugs in synergistic Brij 96 microemulsions: a biophysical
characterization. J. Pharma. Sci. 2015, 104, 22032212.
[26] Kumar, S.; Kumar, N.; Bhanjana, G.; Thakur, R.; Dilbaghi, N. Enhanced Antimicrobial
Activity Of Antibiotics Mixed With Metal Nanoparticles. AIP Confer. Proceed. Amer. Inst.
31
RSC Advances
Page 32 of 35
View Article Online
DOI: 10.1039/C6RA09677H
[32] Kaur, G.; Karir, G.; Mehta S.K. Studies on thermogravimetric analysis and solvophobic
interactionsof micellization of Pd (II) complex in non aqueous solvents. Colloids Surf. A:
[33] Fuguet, E.; R`afols, C.; Roses, M.; Bosch, E. Critical micelle concentration of surfactants
in aqueous buffered and unbuffered systems. Anal. Chim. Acta 2005, 548,95100.
[34] Dutkiewicz, E.; Jakubowska, A. Effect of electrolytes on the physicochemical behavior of
sodium dodecyl sulphate micelles. Colloid Polym. Sci. 2002, 280, 10091014.
[35] Vlachy, N.; Jagoda-Cwiklik, B.; Vacha, R.; Touraud, D.; Jungwirth, P.; Kunz, W.
Hofmeister series and specific interactions of charged headgroups with aqueous ions. Adv.
32
Page 33 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA09677H
[40] Islam, M.M.; Rahman, M.R.; Islam, M.N. Micellization behavior and thermodynamic
properties of n-alkyl trimethylammonium bromide surfactants in aqueous media at different
4, 137143.
[46] Parnsamut, C.; Brimson, S. Effects of silver nanoparticles and gold nanoparticles on IL-2,
IL-6, and TNF- production via MAPK pathway in leukemic cell lines. Genetics Mol. Res. 2015,
14, 3650-3668.
[47] Sulaiman, G. M.; Mohammed, W. H.; Marzoog, T. R.; Al- Amiery, A. A. A.; Kadhum, A.
A. H.; Mohamad, A. B. Green synthesis, antimicrobial and cytotoxic effects of silver
33
RSC Advances
Page 34 of 35
View Article Online
DOI: 10.1039/C6RA09677H
nanoparticles using Eucalyptus chapmaniana leaves extract. Asian Pac. J. Trop. Biomed. 2013, 3,
58-63.
nanoparticles sol against cells of human immune system. Appl. Biochem. Biotechnol. 2015, 176,
817834.
[49] Sulaiman, G.M.; Ali, E.H.; Jabbar, I. I.; Saleem, A. H. Synthesis, characterization,
antibacterial and cytotoxic effects of silver nanoparticles. Digest J. Nanomat. Biostruc.2014, 9,
787 796.
[50] Feng, Q.L.; Wu, J.; Chen, G.Q.; Cui, F.Z.; Kim, T.N.; Kim J.O. A mechanistic study of the
antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J.Biomed.
2015, 3, 9295-9304
34
[48] Barbasz, A.; Owieja, M.; Barbasz, J. Cytotoxic activity of highly purified silver
Page 35 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA09677H
140
120
CH3
H3C
Br
CH3
O3N
Ag
Br
60
20
N
CH3
0
0
Self aggregation
Antimicrobial activity
80
40
CH3
H3C
100
Staphylococcus aureus
35
2
3
Concentration (M)
Escherichia coli
CT-DNA Binding
% Growth inhibition
TABLE PF CONTENTS