R E S EA RCH L E T T E R

Amoebicidal activity of phytosynthesized silver nanoparticlesand their in vitro cytotoxicity to human cells

Hemant P. Borase1, Chandrashekhar D. Patil1, Ismael P. Sauter2, Marilise B. Rott3 & Satish V. Patil1

1School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra, India; 2Programa de P�os-Graduac�~ao em Microbiologia Agr�ıcola e

do Ambiente, Porto Alegre, Rio Grande do Sul, Brasil; and 3Departamento de Microbiologia, Imunologia Parasitologia, Instituto de Ciencias

B�asicas da Sa�ude, Porto Alegre, Rio Grande do Sul, Brasil

Correspondence: Satish V. Patil, School of

Life Sciences, North Maharashtra University,

Jalgaon 425001 Maharashtra, India.

Tel.: +91 257 225 8421;

fax: +91 257 225 8403;

e-mail: satish.patil7@gmail.com

Received 24 May 2013; accepted 4 June

2013. Final version published online 1 July

2013.

DOI: 10.1111/1574-6968.12195

Editor: Simon Silver

Keywords

silver nanoparticles; plant extract;

Acanthamoeba; amoebicidal; cytotoxicity.

Abstract

Acanthamoeba causes infections in humans and other animals and it is impor-

tant to develop treatment therapies. Jatropha curcas, Jatropha gossypifolia and

Euphorbia milii plant extracts synthesized stable silver nanoparticles (AgNPs)

that were relatively stable. Amoebicidal activity of J. gossypifolia, J. curcas and

E. milii leaf extracts showed little effect on viability of Acanthamoeba castellanii

trophozoites. Plant-synthesized AgNPs showed higher amoebicidal activity.

AgNPs synthesized by J. gossypifolia extract were able to kill 74–27% of the

trophozoites at concentrations of 25–1.56 lg mL�1. AgNPs were nontoxic at

minimum inhibitory concentration with peripheral blood mononuclear cells.

These results suggest biologically synthesized nanoparticles as an alternative

candidate for treatment of Acanthamoeba infections.

Introduction

Acanthamoeba is a common protozoa of soil and is fre-

quently found in freshwater and other habitats (Trabelsi

et al., 2012). Cells of Acanthamoeba are usually 15–35 lmin length and oval to triangular in shape when moving.

Cysts are common. Most species are free-living bacteri-

vores, but some are opportunists that can cause infections

in humans and other animals. Acanthamoeba castellanii

can be found at high densities in various soil ecosystems.

It preys not only on bacteria, but also fungi and other

protists. Acanthamoeba castellanii is a facultative pathogen

that has a two-stage life cycle, a growing trophozoite stage

and a dormant cyst stage (Marciano-Cabral & Cabral,

2003). Eradication of Acanthamoeba from an infection

site is often difficult because Acanthamoeba encyst under

unfavorable conditions and the cyst is less susceptible to

anti-amoebic drugs so that disease resurgence occurs after

repetitive therapy that kills trophozoites (Leitsch et al.,

2010). The control of Acanthamoeba infection involves

use of different antimicrobial compounds, namely fluco-

nazole, neomycin, paromomycin, chlorhexidine, hexami-

dine, amphotericin B and chlorhexidine gluconate

(Mathers, 2006). Use of such a variety of compounds

results in post-therapy problems of drug resistance, side

effects and toxicity. Combination therapy is effective in

early stages of infection but becomes ineffective after pro-

longed exposure (Coulon et al., 2010). The high failure

rate of medication may be partially due to poor absorp-

tion of topical anti-amoebic drugs by the thickened sclera

(Seal, 2003) or ineffectiveness of these drugs in killing the

highly resistant cysts and recurrence after stopping of

treatment (Coulon et al., 2010). Plant products could be

used for Acanthamoeba infection treatment as they are

rich sources of bioactive metabolites (Kayser et al., 2003;

Shaalan et al., 2005; Patil et al., 2012). Nanoparticles are

elementary structures of nanotechnology and are impor-

tant materials for fundamental studies and various appli-

cations, including their bioactivities (Patil et al., 2012).

Phytosynthesized silver nanoparticles (AgNPs) as an effec-

tive biological agent have potential over physicochemical

modes of synthesis because of their ecosafety and unique

FEMS Microbiol Lett 345 (2013) 127–131 ª 2013 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

GY

LET

TER

S

synthesis mechanism (Patil et al., 2012). The plants

Jatropha curcas, Jatropha gossypifolia and Euphorbia milii

used in the present study have been reported to have

medicinal application as well as active chemical constit-

uents (Leitsch et al., 2010; Malatyali et al., 2012). Jatro-

pha curcas, J. gossypifolia and E. milii were able to

synthesize AgNPs with higher antimicrobial activity

(Patil et al., 2012). Amoebicidal and cytotoxic capacity

of the AgNPs synthesized from Euphorbian plant

extracts is reported here.

Materials and methods

Preparation of plant extracts

Fresh leaves of plants under study (J. curcas, J. gossypi-

folia and E. milii) were collected from the campus of

North Maharashtra University, Jalgaon, India. Taxo-

nomic identification was done by L. P. Deshmukh,

Department of Botany, JDMVP Science College, Jal-

gaon, India. Leaves were dried and finely ground to

powder. Aqueous extracts were made by mixing 50 g of

plant material with 500 mL water, the contents were

then left for 4 h at 30 °C, filtered through Whatman

no. 1 filter paper, and the filtrate was lyophilized and

stored at 4 °C.

Synthesis of AgNPs

Silver nitrate (100 lg mL�1) was prepared in distilled

water. Lyophilized extract from each plant (10–100 mg)

was added to 25 mL AgNO3 solution in separate vials

and incubated at 37 °C in the dark with constant stirring

for 30 min. During incubation, the color of the solution

changed to yellowish brown, indicating the formation of

AgNPs. This brown solution was used for screening of

amoebicidal activity and further characterization of

AgNPs by UV-Vis spectrophotometry, scanning electron

microscopy (SEM), particle size and zeta potential analy-

sis.

Characterization of AgNPs

Silver nanoparticles were characterized by UV-Vis spec-

trophotometry, particle size analysis and zeta potential

analysis (Figs S1 and S2, Supporting Information).

Scanning electron microscopy

A dried powder sample of AgNPs was mounted on

specimen stubs with double-sided adhesive tape and

coated with gold in a sputter coater (Bal-Tec SCD-050)

and examined under SEM (Philips XL 30), at 12–15 kV

with a tilt angle of 45°.

Test organisms

Acanthamoeba castellanii (strain ATCC 50492) was kept

in PYG medium (2% proteose peptone, 0.2% yeast

extract, 1.8% glucose) at 30 °C. For the experiment,

1 mL of the culture was centrifuged for 5 min at 478 g,

and the pellet washed twice with phosphate-buffered

saline (PBS). The amoebae were suspended in PYG med-

ium at 2 9 104 trophozoites mL�1.

Amoebicidal assay

For the assessment of amoebicidal activity, a 0.1-mL cul-

ture of A. castellanii and 0.1 mL of each test AgNP solu-

tion (50, 25, 12.5, 6.25, 3.125 and 1.56 lg mL�1) were

inoculated in wells of a 96-well U-bottom plate. The plate

was sealed and incubated at 30 °C, monitored on an

inverted microscope and counted in a Fuchs-Rosenthal

counting chamber after 24 h. Viability was assessed using

methylene blue. The experiments were performed in trip-

licate on two different days (n = 6). Test concentrations

of 50, 25, 12.5, 6.25, 3.125 and 1.56 lg mL�1 were

assessed for AgNP samples.

Cytotoxicity assay in peripheral blood

mononuclear cells

Working stock of AgNPs was prepared, and 0.1 mL of two-

fold dilution series of AgNPs was added in a 96-well U-bot-

tom plate by using 10% Roswell Park Memorial Institute

medium. Stimulated peripheral blood mononuclear cells at

2 9 105 per well were added in duplicate to the dilution

suspension and the plates incubated for 5 days at 37 °Cwith humidified 5% CO2 atmosphere. After incubation, cell

viability was determined by (4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide assay (Sigma, St. Louis,

MO). Then, 20 lL (stock, 5 mg mL�1) reagent was added

in each well and incubated at 37 °C for 4 h in a CO2 incu-

bator. Dimethyl sulfoxide (0.1 mL) was added to each well

and kept in the dark for 1 h at room temperature. Optical

density was taken at 550 and 630 nm wavelength, the latter

as a reference wavelength. The assays were performed in

triplicate on 2 different days (n = 6).

Results

Plant-mediated AgNPs were characterized by UV-vis

spectrophotometry (Fig. S1), particle size analysis and

zeta potential (Fig. S2).

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Lett 345 (2013) 127–131Published by John Wiley & Sons Ltd. All rights reserved

128 H.P. Borase et al.

Scanning electron microscopy

Well-dispersed AgNPs with size range of 20–150 nm were

observed by SEM (Fig. 1).

Amoebicidal activity of plant extract and

AgNPs

Amoebicidal activity was investigated of J. curcas,

J. gossypifolia and E. milii plant extracts and AgNPs against

A. castellanii trophozoites. The plant extract solutions

were less effective than the nanosilver products using

plant extract solutions. Activity of extracts from J. gos-

sypifolia, J. curcas and E. milii at 50 lg mL�1 were tested

against A. castellanii trophozoites which showed 22%, 3%

and 3% mortality, respectively, whereas AgNPs synthe-

sized from the same three extracts at 50 lg mL�1 AgNPs

showed greater inhibition (100%, 33% and 5%, respec-

tively; Fig. 2). AgNPs synthesized from J. gossypifolia were

further used at 25, 12.5, 6.2, 3.1 and 1.5 lg mL�1, and

inhibited fewer trophozoites (Fig. 2). The 50% inhibitory

concentration (IC50) of AgNPs of J. gossypifolia found

against A. castellanii was approximately 20 lg mL�1.

Morphology of trophozoites was changed according to

the increase in tested concentrations (Fig. 3). AgNPs, at

all concentration used, were able to prevent encystment

of the trophozoites.

Cytotoxicity of AgNPs synthesized by

J. gossypifolia extract

Silver nanoparticles synthesized using J. gossypifolia

extract were analysed for their cytotoxic nature against

human peripheral blood mononuclear cells. Maximum

inhibition of peripheral blood mononuclear cells was 22%

at 50 lg mL�1 AgNPs (Fig. 4).

Discussion

Silver nanoparticles produced by J. gossypifolia extracts

are potent amoebicidal agents. This may be due to the

small size and stability of AgNPs. As the specific surface

area of nanoparticles is increased, their biological effec-

tiveness can also increase (Sangiliyandi et al., 2009).

Recent studies of antimicrobial activities of AgNPs on

Escherichia coli, Pseudomonas aeruginosa, Staphylococcus

aureus, Staphylococcus epidermidis and Micrococcus luteus

showed cytoplasmic leakage as well as inhibition of

enzymes such as catalase, oxidases and galactosidase

(Sondi & Salopek-Sondi, 2004; Patil et al., 2012). This

Fig. 1. SEM image of Jatropha gossypifolia synthesized AgNPs.

(a) (b)

Fig. 2. (a) Amoebicidal activity on

Acanthamoeba castellanii trophozoites after

treatment with Jatropha gossypifolia, Jatropha

curcas and Euphorbia milii extracts and AgNPs

including AgNO3 as control (all at 50 lg mL�1

Ag). (b) Amoebicidal activity of J. gossypifolia

AgNPs at different concentrations on mortality

of A. castellanii trophozoites. Values are

mean � SD (n = 6).

FEMS Microbiol Lett 345 (2013) 127–131 ª 2013 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

Amoebicidal activity of phytosynthesized silver nanoparticles 129

suggests that Acanthamoba cell death may be occurring

due to involvement of some sort of binding mechanism

or inhibition of important biomolecules of amoeba.

Besides this, AgNPs synthesized from J. gossypifolia were

able to prevent encystment of the trophozoites. In addi-

tion, many plant metabolites such as flavonoids and alka-

loids are reported for their antiparasitic potential

although the mechanism of action is not yet clear

(El-Sayed et al., 2012). Currently available drugs are

known to cause unwanted effects on the plasma mem-

brane of ocular cells (Ehlers & Hjortdal, 2004) thus this

combination of nanoparticles and plant metabolites may

give rise to a broad spectrum of drugs that can be used

in the treatment of Acanthamoba infection. Resistance to

drugs occurs due to the ability of amoeba to turn troph-

ozoites into cysts; compounds that prevent this survival

strategy of A. castellanii can be promising new drugs

against Acanthamoba (Sangiliyandi et al., 2009; Sauter

et al., 2011; Malatyali et al., 2012; Tepe et al., 2012). The

majority of drugs available to treat amoebic infections

display high toxicity for humans, causing side effects,

which often leads to physical damage and even death.

Research is thus being conducted to find alternative

methods for treatment of such infections. In vitro cyto-

toxicity assays in peripheral blood mononuclear cells with

biosynthesized AgNPs showed their nontoxic nature at

the concentrations tested, highlighting the need for fur-

ther studies with in vivo models to develop biologically

well-tolerated treatment of Acanthamoeba infections.

Acknowledgements

We thank Rahul K. Suryawanshi for cytotoxicity tests of

AgNPs, and Dr B.K. Salunke for help with critical revi-

sion of manuscript. H.P.B. acknowledges the Department

of Science & Technology, Government of India, New

Delhi, India, for providing INSPIRE Fellowship (DST/

INSPIRE Fellowship/2011[149]), and C.D.P. acknowledges

the CSIR (09/728(0028)/2012-EMR-I) for the award of a

senior research fellowship.

Authors’ contribution

H.P.B. and C.D.P. contributed equally as first authors on

this manuscript.

(a)

(e) (f) (g)

(b) (c) (d)

Fig. 3. Optical micrographs of Acanthamoeba castellanii during the amoebicidal activity test (24 h) with Jatropha gossypifolia AgNPs. (a) Control,

(b) 1.56 lg mL�1, (c) 3.12 lg mL�1, (d) 6.25 lg mL�1, (e) 12.5 lg mL�1, (f) 25 lg mL�1 and (g) 50 lg mL�1. Nucleus (N), acanthopodia (AC),

cytoplasmic food vacuoles (V), cells with granulation (CG) and cellular fragments (CF) are marked. Cells in the process of degeneration are shown

(e–g). Scale bars = 10 lm.

0

20

40

60

80

100

120

50 25 12.5 6.25 3.12 1.56 0.78 0.39 0

Via

bilit

y (%

)

Concentrations (μg mL–1)

Fig. 4. Cytotoxicity of Jatropha gossypifolia synthesized AgNPs

against human peripheral blood mononuclear cells.

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Lett 345 (2013) 127–131Published by John Wiley & Sons Ltd. All rights reserved

130 H.P. Borase et al.

References

Coulon C, Collignon A, McDonnell G & Thomas V (2010)

Resistance of Acanthamoeba cysts to disinfection treatments

used in health care settings. J Clin Microbiol 48: 2689–2697.Ehlers N & Hjortdal J (2004) Are cataract and iris atrophy

toxic complications of medical treatment of Acanthamoeba

keratitis? Acta Opthalmol Scand 82: 228–231.El-Sayed NM, Ismail KA, Ahmed SAEG & Hetta MH (2012)

In vitro amoebicidal activity of ethanolic extracts of Arachis

hypogaea L., Curcuma longa L., Pancratium maritimum L.

on Acanthamoeba castellanii cysts. Parasitol Res 110:

1985–1992.Kayser O, Kiderlen AF & Croft SL (2003) Natural products as

antiparasitic drugs. Parasitol Res 90: S55–S62.Leitsch D, K€ohsler M, Marchetti-Deschmann M, Deutsch A,

Allmaier G, Duchene M & Walochnik J (2010) Major role

for cysteine proteases during the early phase of

Acanthamoeba castellanii encystment. Eukaryot Cell 9:

611–618.Malatyali E, Tepe B, Degerli S, Berk S & Akpulat HA (2012)

In vitro amoebicidal activity of four Peucedanum species on

Acanthamoeba castellanii cysts and trophozoites. Parasitol

Res 110: 167–174.Marciano-Cabral F & Cabral G (2003) Acanthamoeba spp. as

agents of disease in humans. Clin Microbiol Rev 16: 273–307.Mathers W (2006) Use of higher medication concentrations in

the treatment of Acanthamoeba keratitis. Arch Ophthalmol

124: 923.

Patil SV, Borase HP, Patil CD & Salunke BK (2012)

Biosynthesis of silver nanoparticles using latex from few

euphorbian plants and their antimicrobial potential. Appl

Biochem Biotechnol 107: 776–790.Sangiliyandi G, Kalimuthu K, Ramanathan V, Venkataraman

D, Sureshbabu RP, Jeyaraj M, Nellaiah H & Soo HE (2009)

Biosynthesis, purification and characterization of silver

nanoparticles using Escherichia coli. Colloids Surf B 74:

328–335.

Sauter I, Jaqueline C, Miriam A, Samuel P, Paulo M, Gilsane L

& Rott M (2011) Amoebicidal activity and chemical

composition of Pterocaulon polystachyum (Asteraceae)

essential oil. Parasitol Res 109: 1367–1371.Seal DV (2003) Acanthamoeba keratitis update – incidence,

molecular epidemiology and new drugs for treatment. Eye

17: 893–905.Shaalan EAS, Canyonb D, Younesc MWF, Abdel-Wahaba H

& Mansoura AH (2005) A review of botanical

phytochemicals with mosquitocidal potential. Environ Int

31: 1149–1166.Sondi I & Salopek-Sondi B (2004) Silver nanoparticles as

antimicrobial agent: a case study on E. coli as a model

for Gram-negative bacteria. J Colloid Interface Sci 275:

177–182.Tepe B, Malatyali E, Degerli S & Berk S (2012) In vitro

amoebicidal activities of Teucrium polium and T.

chamaedrys on Acanthamoeba castellanii trophozoites and

cysts. Parasitol Res 110: 1773–1778.Trabelsi H, Dendana F, Sellami A, Sellami H, Cheikhrouhou F,

Neji S, Makni F & Ayadi A (2012) Pathogenic free-living

amoebae: epidemiology and clinical review. Pathol Biol 60:

399–405.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. UV-Visible absorption spectrum of silver nano-

particles synthesized from aqueous extract of Jatropha

curcas (a), Jatropha gossypifolia (b), Euphorbia milii (c)

and 100 lg mL�1 solution of silver nitrate (d).

Fig. S2. (a) Zeta potential distribution and (b) particle

size histogram of AgNPs synthesized using extract from

Jatropha gossypifolia.

FEMS Microbiol Lett 345 (2013) 127–131 ª 2013 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

Amoebicidal activity of phytosynthesized silver nanoparticles 131