Acanthamoeba causes infections in humans and other animals and it is important 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 μg 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.
Acanthamoeba is a common protozoa of soil and is frequently found in freshwater and other habitats (Trabelsi et al., 2012). Cells of Acanthamoeba are usually 15–35 μm in length and oval to triangular in shape when moving. Cysts are common. Most species are free-living bacterivores, 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 fluconazole, neomycin, paromomycin, chlorhexidine, hexamidine, 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 prolonged exposure (Coulon et al., 2010). The high failure rate of medication may be partially due to poor absorption 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 important materials for fundamental studies and various applications, including their bioactivities (Patil et al., 2012). Phytosynthesized silver nanoparticles (AgNPs) as an effective biological agent have potential over physicochemical modes of synthesis because of their ecosafety and unique 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 constituents (Leitsch et al., 2010; Malatyali et al., 2012). Jatropha 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. gossypifolia and E. milii) were collected from the campus of North Maharashtra University, Jalgaon, India. Taxonomic identification was done by L. P. Deshmukh, Department of Botany, JDMVP Science College, Jalgaon, 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 μg 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 analysis.
Characterization of AgNPs
Silver nanoparticles were characterized by UV-Vis spectrophotometry, 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°.
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 medium at 2 × 104 trophozoites mL−1.
For the assessment of amoebicidal activity, a 0.1-mL culture of A. castellanii and 0.1 mL of each test AgNP solution (50, 25, 12.5, 6.25, 3.125 and 1.56 μg 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 triplicate on two different days (n = 6). Test concentrations of 50, 25, 12.5, 6.25, 3.125 and 1.56 μg 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-bottom plate by using 10% Roswell Park Memorial Institute medium. Stimulated peripheral blood mononuclear cells at 2 × 105 per well were added in duplicate to the dilution suspension and the plates incubated for 5 days at 37 °C with 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 μL (stock, 5 mg mL−1) reagent was added in each well and incubated at 37 °C for 4 h in a CO2 incubator. 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).
Plant-mediated AgNPs were characterized by UV-vis spectrophotometry (Fig. S1), particle size analysis and zeta potential (Fig. S2).
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. gossypifolia, J. curcas and E. milii at 50 μg mL−1 were tested against A. castellanii trophozoites which showed 22%, 3% and 3% mortality, respectively, whereas AgNPs synthesized from the same three extracts at 50 μg mL−1 AgNPs showed greater inhibition (100%, 33% and 5%, respectively; Fig. 2). AgNPs synthesized from J. gossypifolia were further used at 25, 12.5, 6.2, 3.1 and 1.5 μg 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 μg 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 μg mL−1 AgNPs (Fig. 4).
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 effectiveness 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 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 addition, many plant metabolites such as flavonoids and alkaloids 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 membrane 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 trophozoites 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 cytotoxicity assays in peripheral blood mononuclear cells with biosynthesized AgNPs showed their nontoxic nature at the concentrations tested, highlighting the need for further studies with in vivo models to develop biologically well-tolerated treatment of Acanthamoeba infections.
We thank Rahul K. Suryawanshi for cytotoxicity tests of AgNPs, and Dr B.K. Salunke for help with critical revision 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), and C.D.P. acknowledges the CSIR (09/728(0028)/2012-EMR-I) for the award of a senior research fellowship.
H.P.B. and C.D.P. contributed equally as first authors on this manuscript.