Colloids and Surfaces B: Biointerfaces 95 (2012) 284–288

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Short communication

Novel route for rapid biosynthesis of copper nanoparticles using aqueous extract
of Calotropis procera L. latex and their cytotoxicity on tumor cells
Shrikant Harne a , Ashwinikumar Sharma b , Mayur Dhaygude b , Shreeram Joglekar c , Kisan Kodam c ,
Manish Hudlikar d,∗
a
Department of Microbiology, University of Pune, Pune 411007, Maharashtra, India
b
Polymer Science and Engineering Division, National Chemical Laboratory, Pashan, Pune 411008, Maharashtra, India
c
Division of Biochemistry, Department of Chemistry, University of Pune, Pune 411007, Maharashtra, India
d
Department of Chemistry, University of Georgia, Athens GA-30605, USA

a r t i c l e i n f o a b s t r a c t

Article history: This paper accounts for novel, low-cost, eco-friendly route for rapid biosynthesis of copper nanoparticles.
Received 19 January 2012 Cysteine proteases present in the latex of Calotropis procera L. were used to fabricate copper nanopar-
Received in revised form 1 March 2012 ticles from copper acetate. Copper nanoparticles were initially characterized by transmission electron
Accepted 12 March 2012
microscopy (TEM) and X-ray diffraction technique (XRD). Transmission electron microscopy (TEM) was
Available online 20 March 2012
used to estimate the size and shape of nanoparticles. The average size of copper nanoparticles was found
to be 15 ± 1.7 nm. Energy dispersive analysis of X-rays (EDAX) showed distinct peaks of copper. Fourier
Keywords:
transform infrared spectroscopy (FTIR) was performed to confirm capping behavior of the latex proteins
Biocompatible
Latex
that contributed to long term stability of copper nanoparticles (6 months) in aqueous medium. Cop-
Copper nanoparticles per nanoparticles synthesized by above method were monodisperse type. Cytotoxicity studies of latex
X-ray diffraction technique (XRD) stabilized copper nanoparticles were carried out on HeLa, A549 and BHK21 cell lines by MTT dye con-
FTIR version assay. HeLa, A549 and BHK21 cells showed excellent viability even at 120 ␮M concentration of
Tumor cells copper nanoparticles. This shows that copper nanoparticles synthesized by above method hold excellent
biocompatibility.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction copper nanoparticles could serve as a potential candidate in follow-

Copper nanoparticles are making significant progress in the
area of nanotechnology and nanomedicine for last ten years
because of their excellent catalytic, optical, electrical and anti-
fungal/antibacterial applications [1,2]. Copper nanoparticles have
been prepared using following methods such as thermal reduction
[3], vacuum vapor deposition [4], microwave irradiation methods
[5], chemical reduction [6], and laser ablation [7]. Polyol method
reported by Park et al. [8] synthesized highly monodispersive cop-
per nanoparticles in air atmosphere.
Biosynthesis of copper nanoparticle was reported by Valodkar
et al. [9] using medicinally important plant Euphorbia nivulia L.
and shown their biological effects on tumor cells. Yoon et al. [10]
and Cioffi et al. [11] have reported the antifungal and bacterio-
static properties of copper nanoparticles recently. This shows that,
∗ Corresponding author. Tel.: +1 478 394 2737.
E-mail addresses: manishhudlikar@gmail.com, manish21@uga.edu
(M. Hudlikar).
ing applications such as nanomedicine, water treatment and food
processing.
Calotropis procera L. has various uses in Indian traditional medic-
inal systems [12,13]. Milky white latex of this plant exhibits diverse
curative properties [14]. Particularly latex of this plant is rich in
protein including anti-oxidant enzymes (AOEs), cysteine protease
with free thiol ( SH) group and tryptophan [15–17].
Present work deals with synthesis of copper nanoparticles at
room temperature using 0.5% aqueous extract prepared from C.
procera L. latex with average diameter of 15 ± 1.7 nm. Biosynthe-
sized copper nanoparticles showed excellent long term stability
in aqueous medium. This show that anti-oxidant enzymes (AOEs),
cysteine protease with free thiol ( SH) group and tryptophan
present in the latex of C. procera L. played important role in reduc-
ing and stabilizing copper nanoparticles. Cytotoxicity studies of
latex stabilized copper nanoparticles were carried out on HeLa,
A549 and BHK21 cell lines by MTT dye conversion assay. HeLa,
A549 and BHK21 cells showed excellent viability even at 120 ␮M
concentration of copper nanoparticles. This shows that copper
nanoparticles synthesized by above method holds excellent bio-
compatibility.

0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2012.03.005
 S. Harne et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 284–288
Fig. 1. Transmission electron micrographs of copper nanoparticles synthesized using (a) 0.1%, (b) 0.3%, and (c) 0.5% aqueous extract prepared from Calotropis procera L. latex.
2. Experimental methods
2.1. Materials and methods
Latex of C. procera L. was collected early in the morning. Crude
latex was obtained by cutting the green stems of C. procera plants.
Milky white latex was stored at −44 ◦ C until use. Copper acetate
analytical grade was purchased from Sigma–Aldrich (USA). All the
aqueous solutions were prepared in distilled de-ionized water.
HeLa, A549 and BHK21 cell lines were obtained from National Cen-
tre for Cell Science (Pune, India) and were cultured and maintained
according to supplier guidelines. 3-(4,5-Dimethylthiazol-2-yl-)-
2,5-diphenyltetrazolium bromide analytical grade was purchased
from Sigma–Aldrich (USA). Eagle’s minimum essential medium
(EMEM) supplemented with 10% fetal bovine serum (FBS) was used
to culture HeLa, A549 and BHK21 cells and was purchased from
Invitrogen (USA).
2.2. Synthesis of copper nanoparticles
In typical reaction mixture, 1 ml crude latex was diluted to
500 ml using distilled deionized water to make it 0.5% and 20 ml
of this latex solution was mixed with 20 ml 3.0 mM aqueous cop-
per acetate solution. Now the mixture was allowed to stand at
room temperature in laboratory ambience with continuous shak-
ing. As soon as, latex solution comes in contact with copper acetate,
spontaneous reduction has occurred within a minute, leading to
synthesis of monodisperse copper nanoparticles.
285
2.3. Characterization of copper nanoparticles
Morphology and size of copper nanoparticles were investi-
gated by transmission electron microscopy (JEOL-1200 EX (TEM
with tungsten electron source) with an accelerating voltage of
120 kV. Powder X-ray diffraction (XRD) was performed using a
X-ray diffractometer (Phillips PW1710, Holland) with CuK␣ radia-
tion  = 1.5405 Å over a wide range of Bragg angles (30◦ ≤ 2 ≤ 80◦ ).
An elemental analysis of the sample was examined by energy
dispersive analyses of X-rays (EDAX) with JED-2300 instrument.
The particle size distribution was measured using a 90 Plus DLS
unit from Brookhaven (Holtsville, USA). Fourier transform infrared
(FTIR) spectroscopic measurements were done using Perkin Elmer
(16 PC-FT-IR) spectrophotometer.
2.4. Cytotoxicity studies
The cytotoxicity studies of latex capped copper nanopar-
ticle were performed on HeLa, A549 and BHK21 cell lines
using 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bro-
mide (MTT) assay following the manufacturer’s protocol. Briefly,
cells were seeded on 96-well plates for 24 h before the assay at
a density of 1.5 × 104 cells/well. Following 24 h growth, medium
was replaced with different concentration of corresponding sample
solutions, which were freshly prepared at varying concentrations
in complete cell culture media. Cells in media containing 10% FBS
with nothing added were used as controls. After 12 or 48 h treat-
ment, cells were rinsed and cultured with fresh medium containing
0.5 mg/ml of MTT for an additional 3 h. Following careful aspiration
286 S. Harne et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 284–288

of MTT solution and media after MTT incubation, different concen-

trations of MTT solubilization solution were added to each well and
thoroughly mixed. The optical density at 570 nm was measured
using a Safire microplate reader (Tecan Systems, Inc., San Jose, CA).
Background absorbance at 690 nm was subtracted. Values were
expressed as a percentage of the control (incubated with media
alone). All conditions were done in sextuplicate in two independent
experiments for each cell line. All experiments were performed in
the aforementioned cell-specific media in a 5% CO2 incubator at
37 ◦ C. The cytotoxicity of copper nanoparticles was assessed by the
MTT (3-[4,5-dimethylthiazole-2-yl]-2, 5-diphenyl tetrazoliumbro-
mide) dye conversion assay [18] and cell viability was expressed as
follows;
Nt
Viability (%) = × 100
Nc

where Nt and Nc are the mean absorbance of copper nanoparticles

treated and control cells respectively (n = 3; where n is the no. of
Fig. 2. Powder X-ray diffraction (XRD) pattern of synthesized copper nanoparticles.
independent experiments).

3. Result and discussion
We found that 0.5% aqueous extract prepared from C. procera
L. latex and 3 mM aqueous solution of copper acetate were opti-
mum for obtaining monodisperse copper nanoparticles (Fig. 1c). A
concentration variation study of copper acetate using 0.5% aque-
ous latex extract was carried out with different concentrations of
copper acetate (1 × 10−3 –5 × 10−3 M). It has been observed that
0.1–0.4% latex solution gave polydisperse copper nanoparticles
(Fig. 1a and b). Copper nanoparticles synthesized using 0.5% aque-
ous extract prepared from latex of C. procera L. and 3 mM copper
acetate under stirring condition at room temperature gave red-
dish solution indicating metallic copper [19]. Copper nanoparticles
were first characterized by high resolution transmission electronic
microscopy (HRTEM) to determine the morphology and size of
the as-prepared copper nanoparticles. As revealed in Fig. 1a and
b, polydisperse spherical particles were obtained. On the other
hand, 0.5% aqueous extract and 3 mM copper acetate solution gave
monodisperse and spherical particles with the sizes ranging from 5
to 30 nm and an average of about 15 ± 1.7 nm (S.D.) were obtained.
Fig. 2 shows X-ray powder diffraction (XRD) patterns of copper
nanoparticles synthesized at room temperature. The XRD pattern is
consistent with earlier reports [3–8]; the peak positions are consis-
tent with metallic copper. All possible peaks of copper are observed,
which indicates the polycrystalline nature of the product. Bragg’s
reflections for copper nanoparticles are observed in XRD pattern
with value of 43.6◦ , 50.7◦ and 74.45◦ representing [1 1 1], [2 0 0]
and [2 2 0] planes of FCC structure of copper [3–8]. The size of the
crystallites was estimated from Debye–Scherrer equation is about
20 nm.
Energy dispersive analysis of X-rays (EDAX) of the synthesized
product gives (Fig. 3a) distinct elemental signals of copper. This
shows that isolated copper nanoparticles possess only metallic
copper without any other impurities. This includes strong ele-
mental peaks at 1.00, 1.50, 2.70 and 8.00 keV. The particle size
distribution was measured using a DLS (dynamic light scattering,
Fig. 3b) unit. Particles formed were monodisperse type. This shows
that monodisperse copper nanoparticles can be fabricated using
enzymes present in the latex of C. procera L. at room temperature.
This simple, eco-friendly and economical route is challenging alter-
native to chemical methods reported earlier [3–8]. Monodispersity
of copper nanoparticle is useful attribute for material scientists to
improve biomedical applications associated with copper nanopar-
ticles [9–14].
The nature of the biomolecules involved in the reduction and
formation of copper nanoparticle was studied by FTIR (Fig. 4) anal-
ysis. FTIR spectra (Fig. 4b) of copper nanoparticles synthesized by
using the latex of C. procera L. shows strong absorption band at

Fig. 3. Energy dispersive spectra (a) of synthesized product with sharp elemental signals of copper. Dynamic light scattering (b) of copper nanoparticles with average diameter
of 15 ± 1.7 nm.
 S. Harne et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 284–288 287

160
3500

Con. of Cu nanoparticles in (uM))

HeLa
(b) 140 A549
3000 BHK 21
120
2916
Transmittance (%T)

2500 100
1027

2000 3423 80
1510 1230
1610 1321 60
1500
40
1000
20
3500
500 (a) 1625 0

0 0 20 40 60 80 100 120 140

4000 3500 3000 2500 2000
-1
Wavenumber (cm )
Fig. 4. FT-IR spectra of the copper nanoparticles before (b) and after 1% sodium
dodecyl sulfate (SDS) detergent treatment to remove the surface protein cap off the
particles (a).
1610 cm−1 . From earlier report we conclude that this frequency is
attributed to binding of NH C O to metals (copper) nanoparticle
[22]. Other significant FTIR bands include 2916 cm−1 (secondary
amine), 1510 cm−1 , 1230 cm−1 , 1321 cm−1 , 1027 cm−1 (amide II,
amide III, carboxylic acid and C N stretching of amines respec-
tively) and alcohol (3423 cm−1 ) clearly implying the presence of
protein on the nanoparticle surface. The proteinaceous material
encapping the particles likely serve as a capping/stabilizing agent
[23–25]. It has been reported that proteins can bind to metal
nanoparticles through the free amine groups or carboxylate ion of
amino acid residues [20,21].
To evaluate if the surface bound capping protein contributes to
the stability, integrity of the nanoparticles and why the FTIR bands
of copper nanoparticles is less than those of latex capped copper
nanoparticles, latex capped copper nanoparticles were treated with
1% sodium dodecyl sulfate (SDS) detergent and heated for 30 min at
85 ◦ C. This treatment resulted in protein denaturation and leads to
an immediate clumping/aggregation of the particles. This is further
supported by the disappearance of most of the bands in the FTIR
spectroscopy (Fig. 4a) including the C H stretch, N H stretch and
carbonyl ( C O C or C O ) stretch vibrations in the amide II
and III linkages. It should be noted that these results not only sup-
port the presence of a protein/peptide encapping the nanoparticle
surface but also demonstrates that the coating can be removed from
the nanoparticle surface. The ability to remove the capping agent is
necessary for applications where no surface coat is required, such
as in particle annealing and thin film formation.
Therefore, the FT-IR spectrum results suggested that anti-
oxidant enzymes (AOEs), cysteine protease and tryptophan [15–17]
with functional groups of amines, alcohols, ketones, aldehydes, and
carboxylic acids might be adsorbed on the surface of the biosynthe-
sized copper nanoparticles using C. procera L. latex.
Cytotoxicity of this latex stabilized copper nanoparticles on
HeLa, A549 and BHK21 cell lines was assessed by the MTT
(3-[4,5-dimethylthiazole-2-yl]-2, 5-diphenyl tetrazoliumbromide)
dye conversion method [18]. Viability test was carried out in
replicates of 3 for each group of cell line. Data was analyzed for
statistical significance using one way analysis of variance (ANOVA)
followed by Bonferroni’s multiple comparison test and results
were expressed as mean ± S.D. using Graph Pad Prism version 3.0
for Windows. After 24 h treatment with filter sterilized copper
nanoparticles on HeLa, A549 and BHK21 cell lines showed excel-
lent viability even upto 120 ␮M concentrations (Fig. 5) of copper
nanoparticles. Therefore, lack of cytotoxicity of latex stabilized
1500 1000 500 (%) Viability of cells
Fig. 5. Cytotoxicity assay – cell viability of HeLa, A549 and BHK-21 cells exposed
to different concentrations (␮M) of copper nanoparticles. Results are expressed as
mean ± S.D. for n = 3 (replicates).
copper nanoparticles on three cell lines suggested that, copper
nanoparticles synthesized using aqueous extract of C. procera L.
latex holds excellent biocompatibility.
4. Conclusion
One step biosynthesis of monodisperse copper nanoparticles
was successfully carried in aqueous solution. Present approach for
synthesizing monodisperse copper nanoparticles using aqueous
extract of C. procera L. latex is important plant based bio-resource
which eliminates the use of synthetic reducing and capping agents
reported in the literature [3–8]. Latex is highly efficient in stabi-
lizing and making these copper nanoparticles biocompatible as
these nanoparticles were non-toxic. These biocompatible copper
nanoparticles can be utilized in developing nanoparticle based plat-
forms for effective drug delivery to induce apoptotic destruction of
tumor/cancer cells.
Acknowledgment
We are grateful to Material Characterization Division, National
Chemical Laboratory (NCL) Pune, India for providing analytical
facilities.
References
[1] A.A. Ponce, K.J. Klabunde, J. Mol. Catal. A 225 (2005) 1.
[2] Z. Huang, F. Cui, H. Kang, J. Chen, X. Zhang, C. Xia, Chem. Mater. 20 (2008) 5090.
[3] N.A. Dhas, C.P. Raj, A. Gedanken, Chem. Mater. 10 (1998) 1446.
[4] Z. Liu, Y. Bando, Adv. Mater. 15 (2003) 303.
[5] Y. Zhao, J.J. Zhu, J.M. Hong, N. Bian, H.Y. Chen, Eur. J. Inorg. Chem. 20 (2004)
4072.
[6] M. Yang, J.J. Zhu, J. Cryst. Growth 256 (2003) 134.
[7] M.S. Yeh, Y.S. Yang, Y.P. Lee, H.F. Lee, Y.H. Yeh, C.S. Yeh, J. Phys. Chem. B 103
(1999) 6851.
[8] B.K. Park, S. Jeong, D. Kim, J. Moon, S. Lim, J.S. Kim, J. Colloid Interface Sci. 311
(2007) 417.
[9] M. Valodkar, N.J. Ravirajsinh, C.T. Menaka, V.D. Ranjitsinh, S. Thakore, Mater.
Chem. Phys. 128 (2011) 83.
[10] K. Yoon, J.H. Byeon, J. Park, J. Hwang, Sci. Total Environ. 373 (2007) 572.
[11] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, Chem. Mater.
17 (2005) 5255.
[12] Z. Iqbal, M. Lateef, A. Jabbar, G. Muhammad, M.N. Khan, Ethanopharmacology
14 (2005) 256.
[13] S.C. Jain, R. Sharma, R. Jain, R.A. Sharma, Fitoterapia 3 (1996) 275.
[14] M.V. Ramos, V.C. Aguiar, V.M.M. Melo, R.O. Mesquita, P.P. Silvestre, J.S. Oliveira,
R.S.B. Oliveira, N.M.R. Macedo, N.M.N. Alencar, Ethanopharmacology 11 (2007)
115.
[15] C.D.T. Freitas, J.S. Oliveira, M.R.A. Miranda, Plant Physiol. Biochem. 45 (2007)
781.
[16] K.I. Abraham, P.N. Joshi, Biochim. Biophys. Acta 568 (1979) 120.
[17] G. Pal, N.K. Sinha, Arch. Biochem. Biophys. 202 (1980) 321.
288 S. Harne et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 284–288

[18] T. Mossman, J. Immunol. Methods 65 (1983) 55.
[19] S.N. Masoud, D. Fatemeh, M. Noshin, Polyhedron 27 (2008) 3514.
[20] G.W. Jeong, Y.W. Lee, M. Kim, S.W. Han, J. Colloid Interface Sci. 97 (2009) 329.
[21] A. Ahmad, S. Senapathi, M.I. Khan, R. Kumar, M. Sastry, Langmuir 19 (2003)
3550.
[22] Z. Lin, J. Wu, R. Xue, Y. Yong, Spectrochim. Acta A 61 (2005) 761.
[23] S.K. Das, A.R. Das, A.K. Guha, Langmuir 25 (2009) 8192.
[24] A.K. Suresh, D.A. Pelletier, W. Wang, J.W. Moon, B. Gu, N.P. Mortensen, D.P.
Allison, D.C. Joy, T.J. Phelps, M.J. Doktycz, Environ. Sci. Technol. 44 (2010)
5210.
[25] S.A. Kumar, M.K. Abyaneh, S.W. Gosavi, S.W. Kulkarni, A. Ahmad, M.I. Khan,
Biotechnol. Appl. Biochem. 47 (2007) 191.