Rapid synthesis for monodispersed gold
nanoparticles in kaempferol and anti-leishmanial
efficacy against wild and drug resistant strains
Asim Halder,
a
Suvadra Das,
a
Tanmoy Bera
b
and Arup Mukherjee
*
a
Leishmaniasis is a WHO notified neglected tropical disease (NTD) caused by the Leishmania parasite
species. The parasite locates rapidly in the macrophage cells and survives there for a long period of time
with or without any symptomatic response. Eliminating Leishmania from the macrophage sites is
complicated. Only a few antileishmanial drugs are known and most of them have developed resistance
over the time. We have synthesized highly monodispersed gold nanoparticles (KAunp) in a rapid
reduction reaction catalyzed in kaempferol. Kaempferol stabilized nanoparticles in 18.24 nm size domain
were used as leishmanicidals in macrophage infested conditions. KAunps' CC
50
in the macrophages was
at 2.4 10
2
mM while that for sodium stibogluconate was 27 mM. Phagocytic uptake in infested cells was
fast and remarkable. KAunps were explicitly effective against both the wild and drug resistant organisms.
Monodispersed gold nanoparticles synthesized in green technology appeared as safer alternatives for
leishmanial chemotherapy.
1. Introduction
Gold nanoparticles are veritable tools for applications in the
chemical biology interfaces.
1
Particle parameters like the size,
morphology, scaffolding, surface charge and dispersity are
some of the very crucial parameters for successes in biology.
Nanoscale surface chemistry and particle aspect ratio for
example inuences cellular trafficking.
2,3
In sharp contrast to
the so polymer nanoparticles, inexible nanocarriers like the
metal and metal oxides face much faster uptake in infected cell
targets.
4,5
Gold nanoparticles near spherical were apparently
safe and elicit low or no systemic immunity reactions.
Furthermore, particles in sub 100 nm ranges were encourag-
ingly effective in cancer chemotherapy and microbial infection
control.
6,7
Nanoparticles by design were also projected as one
successful strategy against complex immune cell resident
infections like that in HIV aids and tuberculosis.
8–10
We postu-
lated that avonoid stabilized spherical gold nanoparticles may
provide some useful tools for precise control of macrophage
resident leishmania infections.
Synthesis of gold nanoparticles typically involved reduction of
tetrachloroauric acid in sodium borohydride or sodium citrate.
Unfortunately, the temperature dependent citrate method
produced quality particles only up to 50 nm in size and beyond
which they are polydispersed and nonspherical.
11
Sodium boro-
hydride reduction is very fast and particle uniformity is always
affected. Various green techniques for nano-gold synthesis and
in situ functionalization were experimented.
12
Inconsistent
particle characteristics reported in similar conditions was mostly
due to sluggish redox reactions. Reducing gold onto seeds prior
to the growth steps is perhaps a practicable strategy for consis-
tent nanoparticle characteristics.
13
However, scale up synthesis
and appropriate bio-molecular functionalizations were not oen
very feasible in similar techniques. Furthermore, reagents that
are frequently used for gold nanoparticles stabilization are
unsafe and not oen biocompatible. For example, gold nano-
particles dispersion stability was oen achieved with strongly
bound toxic passivaters like CTAB (cetyltrimethylammonium
bromide). Stable aqueous dispersion of uniform size gold nano-
colloid carrying no toxic debris is nevertheless essential for
therapeutic applications. Non-thermal gold nanoparticle
synthesis and reliable particle quality for therapeutic application
however remained elusive for a long time.
Leishmaniasis is a vector born neglected tropical disease
(NTD). The disease is caused by Leishmania parasite species. The
infection is endemic in different tropical regions. The organism
is also known to migrate rapidly in war torn regions around the
globe. The disease is now a major public health threat. Among
all types, the visceral leishmaniasis (VL) caused by Leishmania
donovani is life threatening and is most difficult one to be
controlled. VL asymptotic infections are quite common which
oen surfaces later in immune compromise conditions like that
in case of HIV-aids. Currently, there exists no perfect stratagem
to contain similar infections in connement. Once infested, the
a
Department of Chemical Technology, University of Calcutta, 92, A.P.C. Road,
Kolkata-700 009, West Bengal, India. E-mail: arupm1234@gmail.com; Fax:
+913323519755; Tel: +913323508387
b
Department of Pharmaceutical Technology, Jadavpur University, 188 Raja S. C. Mullik
Road, Kolkata-700 032, West Bengal, India
Cite this: RSC Adv.,2017,7,14159
Received 23rd December 2016
Accepted 17th February 2017
DOI: 10.1039/c6ra28632a
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parasite quickly locates themselves into phagocytic macrophage
cells and employs biochemical strategies to survive and
multiply.
14
Drug resistance is rampant and old drugs like
sodium stibogluconate are no longer effective. Amphoterecin B
(AmB) is effective but expresses a very narrow therapeutic
window and is forbiddingly costly. Compounds like paromo-
mycin or miltefosine are highly toxic and even teratogenic in
some cases. Cross resistance in combination therapy is also very
common.
15,16
Metal nanoparticles are explicitly experimented in
therapeutics albeit a lot of incoherency in particle properties.
Hard nanoparticles by design are also in high demand for
infectious disease control and swi therapeutic delivery. Biosafe
highly monodispersed noble metal nano-spheroids as drug
carriers were considered as an early solution against immune
cell resident infections.
17
This work was planned to address
twine objectives of bio-safe gold nanoparticles synthesis in
small size regime and containment of VL infections in micro-
phage infested conditions. Antioxidant polyphenol kaempherol
was used for the rst time for uniform size gold nano-scaffolds
synthesis in low temperature conditions. Kaempherol and the
kaempherol stabilized nano-gold were used further as leish-
manicidals in macrophage resident conditions.
2. Experimental
2.1. Materials
Chloroauic acid (HAuCl
4
$3H
2
O), kaempferol, Amphotericin B
(AmB) and RPMI-1640 cell culture medium were all purchased
from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade solvents
and water were procured from E. Merck or Spectrochem (Mumbai,
India). Sodium stibogluconate (SSG) was a kind gi from Albert
David Ltd. (Kolkata, India). Standard glasswares of Borosil®
(Mumbai, India) make were used for preparation and analysis
experiments. All other reagents used were of analytical grade.
2.2. Synthesis of gold nanoparticles
Gold nanoparticles were synthesized under low temperature
sonication in kaempferol. In a typical procedure, 3 ml of 1.0 mM
chloroauric acid in water was added with 1 ml of 4 mM
kaempferol and was vortexted to equilibrate. The reaction
mixture was put to sonication for 1 min in a 50 Hz bath soni-
cator (UD 100SH-3L Takashi, Japan) maintained at 4
C. KAunps
were collected aer centrifugation at 15 000 rpm for 30 min. The
nanoparticles were washed in water, re-centrifuged and nally
diluted in water and stored at 4
C for further applications.
2.3. Characterization of nanoparticles
Formation of nanoparticles was conrmed by UV-vis spectral
analysis. The plasmonic absorbances were monitored in
a double beam UV-vis spectrophotometer (Shimadzu Japan-
2550) at different reaction stages. The samples were diluted
with water and absorption spectra were recorded at medium
speed in the wavelength range of 200–800 nm. The particle size
in hydrodynamics was monitored in a zetasizer Nano ZS (Mal-
vern Instruments, Malvern, UK). A 4 mW He–Ne laser beam was
used at 633 nm and back scattering angle of 173
. Particle
polydispersity index (PDI) was recorded in the same instrument.
Analysis experiments were conducted in triplicate for each
sample batch and the results were reported as the mean
standard deviation. Disposable zeta cells were used to measure
particle electrophoretic mobility and Smoluchowski equation
was applied for determining KAunp zeta potential (z) in water
medium. A JEOL-JEM 2010 (JEOL, Tokyo, Japan) high-resolution
transmission electron microscope (HR-TEM) operated at 200 kV
and equipped with an energy-dispersive X-ray (EDAX) device was
used to measure the size and homogeneity of the synthesized
particles. Samples were prepared by applying 30 ml of diluted
nanoparticle solution onto carbon-coated copper TEM grids (300
mesh, Ted pella, Redding, CA, USA). Excess solution was
removed with a lter paper and the grids were allowed to dry
prior to measurements. Approximately, 150 particles were
counted and were measured for a size distribution study.
Elemental analyses of the residues were carried out in EDAX
analyzer attached to the TEM system. FT-IR studies were con-
ducted over a wave number range of 4000 to 400 cm
1
in FT/IR-
670 plus (Jasco, Tokyo, Japan) using DLATGS detectors. Pressed
KBr disks were used at 4 cm
1
resolution. Both KAunp and
kaempferol were studied and the data stacked in Bio-Rad Kno-
wItAll (Bio-Rad, Hercules, CA) soware for comparative analysis.
X-ray diffraction analysis was performed on a Bruker AXS D8
(Bruker, Karlsruhe, Germany) diffractometer with Cu ka radia-
tion (l ¼ 1.5405
˚
A) operated at an accelerating voltage of 40 kV
and current intensity 40 mA. The scanning rate applied was
0.020 s
1
in the 2q ranges of 30–80
. KAunp samples were
digested in aqua regia for 2 h and Elan-DRC-e (Parkin Elmer,
USA) ICPMS facility was used for Au analysis. Instrument oper-
ating conditions were 1050 Hz radio-frequency forward power,
12.8 l min
1
plasma carrier gas ow (argon), and 26 rpm nebu-
lizer pump speed. Gold concentration was determined using
Au
197
isotope levels. Calibration curve was developed from the
instrument standards in concentration range for 50 to 1000 ppb.
2.4. In vitro assessment of antileishmanial activity
2.4.1. Parasites and cell line. L. donovani promastigotes AG
83(MHOM/IN/1983/AG83), isolated from a ‘kala-azar’ patient
was received as a gi from the Indian Institute of Chemical
Biology, Council of Scientic and Industrial Research, Kolkata,
India and used for experiments.
18
L. donovani amastigotes were
grown and maintained in MAA/20 medium at pH 5.5 in 37
2
C incubator with 5% v/v CO
2
/air mixture. In a span of 120 h
promastigotes differentiated to amastigotes and cultures
were maintained by dilutions once in a week. Mouse (swiss
albino, female 22 2 g) peritoneal macrophages were collected
aer 5 days of an i.p injection of 0.5 ml 4% thioglycollate broth.
Exudate cells were harvested in RPMI-1640 medium supple-
mented with 2 g l
1
NaHCO
3
, 10% fetal calf serum, 100 U ml
1
penicillin and 50 mgml
1
streptomycin. Aer incubation for 2 h
at 37 2
C, non-adherent cells were removed by thorough
washing and adherent macrophages were cultured for 24 h at 37
2
C. Cultures prepared contained $95% of macrophages
were evaluated by their ability to take up latex bead and by
morphology. All animal studies were performed in compliance
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with the guidelines of Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA),
Government of India and the experimental protocols were
approved by the institutional animal ethical committee (IAEC)
of Jadavpur University (AEC/PHARM/1601/12/2016).
2.4.2. Macrophage infested resistant amastigote. Resis-
tance development in L. donovani AG83 promastigotes were rst
performed by addition of SSG or paromomycin (PMM) in the
growth medium at an increasing concentration.
19
Promasti-
gotes in the logarithmic growth phase were cultured continu-
ously at a particular drug concentration which killed 90 to 95%
of the parasites exposed. Surviving subpopulation of parasites
that were stabilized to grow successfully in new drug concen-
tration was further exposed to subsequent higher concentra-
tions of SSG or PMM. Drug resistant phenotypes were
differentiated into amastigotes within 120 h of growth in MMA/
20 medium at pH 5.5 placed in a 5% v/v CO
2
/air mixture and
37 2
C at incubator.
2.4.3. Anti-leishmanial assay in vitro. Anti-amastigote
activities were studied by direct counting growth inhibition
assay with slight modications.
20
Amastigotes were seeded at an
initial concentration equivalent to early log phase (3 10
5
amastigotes per ml) and multiplied for 72 h either in the
medium alone or in presence of serial dilution of the test
compound until the late log phase (1 10
6
cells per ml). Axenic
amastigote number doubled three to four times during the
assay in maintenance media. IC
50
(50% inhibitory concentra-
tion) for each test compound was determined in parallel. The
amastigotes were counted using hemocytometer slides under
a microscope. All experiments were done in triplicate for
comparative purposes.
2.4.4. Anti-amastigotes activity in macrophage. Suscepti-
bility of L. donovani amastigotes in macrophages was evaluated
as proposed by Chang et al. with minor modication.
21
Briey
macrophages were seeded in a-10 medium at 4 10
4
cells per
well count in chamber slides and kept for 24 h at 37 2
C for
attachments. Macrophages were infected to axenic amastigotes
(4 10
5
amastigotes per well) for 4 h at 37 2
C. To remove
non-phagocytosed parasites slides were washed with Dulbecco's
phosphate-buffer saline. Standard drug and test compounds
were dissolved in culture medium and incubated with different
concentrations for 72 h at 37 2
C in a 5% v/v CO
2
/air mixture
in incubator. Treatment solutions and the medium were
replaced with fresh ones every 24 h. Control wells of the infected
macrophages were incubated with the same medium. Activity in
each case was estimated from the percentage reduction in
amastigote number in treated and untreated cultures. Obser-
vation were recorded in methanol-xed, giemsa stained (10%
v/v) preparations. The antileishmanial effects were expressed as
IC
50
values and the results were averaged in each concentration
from triplicate observations.
2.5. Cytotoxicity studies against mammalian macrophages
Macrophage cells were cultured in minimum essential medium
(MEM, Gibco), supplemented with 20 mM
L-glutamate, 16 mM
NaHCO
3
, 5% fecal calf serum and penicillin–streptomycin. The
assay was performed in 96-well tissue culture plates in presence
of standard counts of the macrophages. The wells were seeded
with test solutions in different dilution and the viable macro-
phages were counted microscopically aer 48 h of exposure at
37 2
C in a 5% v/v CO
2
incubator.
2.6. Nanoparticle macrophage uptake studies in TEM
The cellular uptake of KAunps into macrophage cells was
observed in TEM (FP 5018/40 (TECHNAI G
2
SPIRIT BioTWIN,
Czech Republic). Macrophage cells were cultured on 6 well
tissue culture plates and incubated overnight for growth under
5% CO
2
at 37 2
C. In the next day, growth plates were exposed
to 10 mM KAunp concentration for different time periods
(10 min, 30 min, 1 h and 2 h). Cells aer exposure were washed
with 0.1 M PBS and pellets were spun at 3000 rpm for 10 min.
Washed cells were collected aer three similar repeat centrifu-
gations and were xed in a mixture of 2% paraformaldehyde
and 2.5% glutaraldehyde in 0.1 M PBS for 2 h.
22
Fixed samples
were washed and stained with 1% osmium tetra oxide at 4
C for
observations. Cell pellet dehydration was performed in graded
concentration of acetone (30, 50, 70, 90, and 100%) followed by
toluene. Samples were subsequently embedded in epoxy resin
and cut into 80 nm thin sections and analyzed in TEM at 75 kV.
2.7. Statistical analysis
Data collected were expressed as the mean standard devia-
tion. Biological analysis data were gathered in triplicates and
were assessed following Student's t test. Statistical signicance
was recorded when p < 0.05.
3. Results and discussion
3.1. Gold nanoparticle synthesis
The size and purity of gold nanoparticles in ab initio synthesis
depends generally on nucleus formation reduction rate, and
subsequent molecular association. Turkevich originally re-
ported citrate stabilized 20 nm gold nanoparticles synthesis in
water.
13
Particle size control by varying reagent ratio was later
achieved and the reduction kinetics studied by different
researchers.
11,12,23,24
In our case gold nanoparticles were
synthesized by reducing HAuCl
4
with kaempferol under ultra-
sound in low bath temperature conditions. The polyphenolic
compound kaempferol is a powerful reducing agent (reduction
potential 0.98 V).
25
Fast reduction of HAuCl
4
under constant
frequency ultrasound at 4
C was presented in Fig. 1. The
solution color changed from pale yellow to violet within 15 s.
Plasmonic intensity at 541 nm gained rapidly thereaer and
a saturation point was achieved within 1 min. Fern's nucleation-
diffusion and growth mechanism appeared responsible for
nanoparticles synthesis in low temperature ultrasound condi-
tions.
26
This reaction when monitored at different time points
the plasmonic peak was always recorded at 541 nm up to 1 min
of reaction time (Fig. 2A). Beyond that time the peak intensity
was reduced and a peak shi was observed due to particle
aggregation and irregular formations (Fig. 2B). The plasmonic
absorbance peak decreased when the reactive molar
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concentration was adjusted to the lower ratios (4 : 1 > 3 : 1 >
2 : 1 > 1 : 1). The peak responsible for the aqueous gold salt
solution (Fig. 1B) disappeared completely and the intense violet
color xed peak due to KAunp soon appeared (Fig. 1A). The
avonoid kaempherol and chloroauric acid molar ratio for
ultrasound reduction reaction was therefore set at 4 : 1. When
HAuCl
4
was exposed to kaempferol without sonication no color
change was recorded even aer 2 h of observation period
(Fig. 2A inset). The Au
3+
Au
0
standard reduction potential is
1.0 V.
27
At low temperature conditions kaempherol only has
failed to initiate Au nuclear transformations. In conducting
medium like water ultrasound is known to create pressure
waves and induce molecular vibrational motions. Ultrasound
compression waves generate cavities consecutively inside the
bulk medium. Short lived water bubbles developed due to cav-
itational effects expanded under negative pressure excursion
Fig. 1 (A) UV-vis spectra of gold nanoparticles prepared in different molar proportion of kaempferol and gold chloride: (a) 1 : 1, (b) 2 : 1, (c) 3 : 1,
(d) 4 : 1, (e) 5 : 1, (f) 6 : 1and (g) 7 : 1. (B) UV-vis spectra of HAuCl
4
aqueous solution. Inset images taken from aqueous gold salt solution (inset B)
and gold nanoparticles (inset A).
Fig. 2 UV-vis spectra obtain from different time point of reaction: (A) spectra recorded at (a) 5 s, (b)10 s, (c)15 s, (d) 20 s, (e) 30 s, (f) 40 s, (g)
50 s and (h) 60 s. inset figure, UV-vis spectra recorded without sonication. (B) spectra recorded at (i) 70 s, (j) 80 s, (k) 90 s, (l) 100 s, (m) 110 s
and (n) 120 s.
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and crush very fast. The energy release per minute due to cav-
itational implodes in bubble centers is extremely high.
28
The
energy exposure on kaempferol molecules under ultrasound
has thus enabled a rapid reduction, gold nucleation and highly
monodispersed nanoparticles formations. When ultrasound
induced reduction reaction was continued beyond 1 min gold
nanoparticles faced enhanced cavitational collapse, fast colli-
sion and likely overgrowth.
Kaempferol is a avonoid with highest antioxidant capacity
among all plant polyphenolic compounds. The hydroxyl group
in avonoid ring B played an important role in the hydrogen
atom transfer reactions.
29
The rate of reaction in kaempferol
with one hydroxyl group in ring B was faster than quercetin
having 3
0
,4
0
-OH groups. Furthermore, the 3-OH substitution in
the ring C and the torsion angle for ring B and C provided
a crucial stability parameter for the kaempferol free radical. The
Scheme 1 Schematic diagram showing possible mechanism of gold nanoparticles formation and stabilization in kaempferol under sonication.
Fig. 3 (A) TEM study of KAunp. (B) KAunp size distribution histogram. (C) SAED pattern. (D) High resolution image of single nanoparticle. (E) EDAX
spectrum of KAunp.
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