Parasitol Res DOI 10.1007/s00436-015-4556-2

ORIGINAL PAPER

Green-synthesized silver nanoparticles as a novel control tool against dengue virus (DEN-2) and its primary vector Aedes aegypti Vasu Sujitha 1 & Kadarkarai Murugan 1 & Manickam Paulpandi 1 & Chellasamy Panneerselvam 1 & Udaiyan Suresh 1 & Mathath Roni 1 & Marcello Nicoletti 2 & Akon Higuchi 3 & Pari Madhiyazhagan 1 & Jayapal Subramaniam 1 & Devakumar Dinesh 1 & Chithravel Vadivalagan 1 & Balamurugan Chandramohan 1 & Abdullah A. Alarfaj 4 & Murugan A. Munusamy 4 & Donald R. Barnard 5 & Giovanni Benelli 6

Received: 20 May 2015 / Accepted: 25 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Dengue is an arthropod-borne viral infection mainly vectored through the bite of Aedes mosquitoes. Recently, its transmission has strongly increased in urban and semi-urban areas of tropical and sub-tropical regions worldwide, becoming a major international public health concern. There is no specific treatment for dengue. Its prevention and control solely depends on effective vector control measures. In this study, we proposed the green-synthesis of silver nanoparticles (AgNP) as a novel and effective tool against the dengue serotype DEN-2 and its major vector Aedes aegypti. AgNP were synthesized using the Moringa oleifera seed extract as reducing and stabilizing agent. AgNP were characterized using a variety of biophysical methods including UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray

spectroscopy (EDX), X-ray diffraction (XRD), and sorted for size categories. AgNP showed in vitro antiviral activity against DEN-2 infecting vero cells. Viral titer was 7 log10 TCID50/ml in control (AgNP-free), while it dropped to 3.2 log10 TCID50/ml after a single treatment with 20 μl/ml of AgNP. After 6 h, DEN-2 yield was 5.8 log10 PFU/ml in the control, while it was 1.4 log10 PFU/ml post-treatment with AgNP (20 μl/ml). AgNP were highly effective against the dengue vector A. aegypti, with LC50 values ranging from 10.24 ppm (I instar larvae) to 21.17 ppm (pupae). Overall, this research highlighted the concrete potential of greensynthesized AgNP in the fight against dengue and its primary vector A. aegypti. Further research on structure–activity relationships of AgNP against other dengue serotypes is urgently required.

* Giovanni Benelli [email protected]; [email protected]

Keywords Botanical insecticides . Mosquito-borne diseases . Moringa oleifera . silver nanoparticles . Aedes aegypti . cytotoxicity

1

Division of Entomology, Department of Zoology, School of Life sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India

2

Department of Environmental Biology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy

3

Department of Reproduction, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan

4

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

5

Center for Medical, Agricultural, and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608, USA

6

Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy

Introduction Dengue is a mosquito-borne viral disease transmitted by female mosquitoes, mainly Aedes aegypti and, to a lesser extent, Aedes albopictus. Recently, dengue transmission has strongly increased in urban and semi-urban tropical areas worldwide, becoming a major international public health concern. Over 2.5 billion people are now at risk from dengue. The World Health Organization estimates that there may be 50–100 millions of dengue infections worldwide every year. There are four distinct, but closely related, serotypes of the virus that

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cause dengue (DEN-1, DEN-2, DEN-3, and DEN-4). Recovery from infection by one provides lifelong immunity against that particular serotype. However, cross-immunity to the other serotypes after recovery is only partial and temporary (WHO 2012). Currently, there is no specific treatment for dengue, even if the development of a vaccine is in progress (Murrell et al. 2011; WHO 2015). Its prevention and control solely depends on effective vector control measures (Suresh et al. 2015; WHO 2015). Aedes larvae and pupae are usually targeted using organophosphates and insect growth regulators. Indoors residual spraying and insecticide-treated bed nets are also employed to reduce transmission of malaria in tropical countries. However, synthetic chemicals have strong negative effects on human health and the environment and induce resistance in a number of mosquito species (Hemingway and Ranson 2000). In this scenario, eco-friendly control tools are urgently needed. In the latest years, huge efforts have been carried out to investigate the efficacy of botanical products against mosquito vectors; many plant-borne compounds have been reported as effective against Culicidae, acting as adulticidal, larvicidal, ovicidal, oviposition deterrent, growth and/or reproduction inhibitors, and/or adult repellents (e.g., Amer and Mehlhorn 2006a, b; Panneerselvam et al. 2012; Benelli et al. 2015a, b). Nanobiotechnologies have the potential to revolutionize a wide array of applications, including drug delivery, diagnostics, imaging, sensing, gene delivery, artificial implants, tissue engineering, and pest management (Elechiguerra et al. 2005). The plant-mediated biosynthesis (i.e., Bgreen synthesis^) of metal nanoparticles is advantageous over chemical and physical methods, since it is cheap, single-step, does not require high pressure, energy, temperature, and the use of highly toxic chemicals (Goodsell 2004). A growing number of plants and fungi have proposed for efficient and rapid extracellular synthesis of silver and gold nanoparticles (see Rajan et al. 2015 for a recent review), which showed excellent mosquitocidal properties, also in field conditions (e.g., Rajakumar and Rahuman 2011; Dinesh et al. 2015; Suresh et al. 2015; Murugan et al. 2015a, b). In this study, we proposed the green-synthesis of silver nanoparticles (AgNP) as a novel and effective tool against the serotype DEN-2 and its major vector A. aegypti. AgNP were synthesized using the Moringa oleifera (Moringaceae) seed extract as reducing and stabilizing agent. AgNP were characterized using a variety of biophysical methods including UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energydispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and sorted for size categories. Green-synthesized AgNP were tested for antiviral activity against the dengue serotype DEN-2 using vero cell culture assays and growth inhibition tests. Furthermore, M. oleifera seed extract and green-synthesized AgNP were tested for larvicidal and

pupicidal toxicity against the primary dengue vector A. aegypti.

Materials and methods Study species and collection of plant material M. oleifera is a small, fast-growing tree with an ancient tradition in ethno-pharmacology. Its plant parts are rich in flavonoids, tannins, saponins, terpenoids, proanthocyanadins, and cardiac glycosides (Vinoth et al. 2012). M. oleifera seeds have been studied for their anti-microbial, anti-tumor, anti-inflammatory, antispasmodic, and diuretic properties (Cárceres et al. 1992; Guevara et al. 1999; Bharali et al. 2003; Ali et al. 2004; Chuang et al. 2007). In this study, the seeds of M. oleifera were collected from the campus of the Bharathiar University (Coimbatore, India). Seeds were identified by an expert taxonomist at the Department of Botany (Bharathiar University, Coimbatore). Voucher specimens were stored in our laboratories and are available under request. M. oleifera-mediated biosynthesis of silver nanoparticles M. oleifera seeds were washed carefully with distilled water and shade-dried at room temperature (28±2 °C) for 2 days; 20 g of seeds were powdered using an electrical blender; 5 g of seed powder were added in a 300-mL Erlenmeyer flask filled with 100 mL of sterilized distilled water, then the mixture was boiled for 5 min, before finally decanting it. The seed extract was filtered using Whatman filter paper no. 1, stored at −4 °C and tested within 5 days. The filtrate was treated with aqueous 1 mM AgNO3 solution in an Erlenmeyer flask, and incubated at room temperature. Silver nitrate was purchased from the Precision Scientific Co. (Coimbatore, India). A dark brown solution indicated the formation of AgNP, since aqueous silver ions were reduced by the M. oleifera seed extract generating stable Ag0 nanoparticles. Characterization of green-synthesized silver nanoparticles Following the methods reported by Murugan et al. (2015a, b), the biosynthesis of AgNP was confirmed by sampling the reaction mixture at regular intervals (2 ml per sampling). The maxima absorption was scanned by UV–vis spectroscopy at wavelength of 200–600 nm, using a UV-3600 Shimadzu spectrophotometer with 1-nm resolution. Furthermore, the reaction mixture was subjected to centrifugation at 15,000 rpm for 20 min. The resulting pellet was dissolved in de-ionized water and filtered through Millipore filter (0.45 μm). An aliquot of this filtrate containing AgNP was used for SEM, EDX, FTIR, and XRD. SEM studies were carried out using a FEI

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QUANTA-200 SEM. EDX analyses were conducted using a JEOL-MODEL 6390. XRD analysis of AgNP-coated glass substrates was carried out on a Phillips PW1830 instrument operating at 40 kV and current of 30 mA with Cu Kα radiation. FTIR measurements were carried out using a PerkinElmer Spectrum 2000 FTIR spectrophotometer. The AgNP size distribution was determined using the particle analyzer Malvern Nanosizer (Malvern Instrument, UK), where the size was analyzed measuring size-dependent fluctuation of a scattering laser light on AgNP. Cells and virus C6/36 and vero cells (i.e., originally propagated from African green monkey kidney cells) were purchased from the National Centre for Cell Sciences (NCCS, Pune, India). Both cell lines were maintained and propagated in Eagle’s minimum essential medium (EMEM) containing 10 % fetal bovine serum. Cultured C6/36 and vero cells were incubated at 28 and 37 °C, respectively, in 5 % CO2 humidified chamber. For virus propagation, the serum concentration of the medium was reduced to 2 %. Dengue virus type-2 (DEN-2) New Guinea C strain was propagated using C6/36 cell line, and harvested after cytopathic effect (CPE) presentation 7 days post-infection. After titration, viral stock was maintained at −70 °C. Cytotoxicity assays In citotoxicity assays, quadruplicate wells of confluent monolayers of vero cells were grown in 96-well tissue culture plates. Cells were incubated with different concentrations of AgNP. Then, we examined cell viability, as the ability of the cells to cleave the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2ol)-2,5diphenyltetrazoliumbromide), Sigma Chem. Co. St. Louis, USA], by the mitochondrial enzyme succinate dehydrogenase which develops a formazan crystal. Each concentration was replicated three times. Treatment of viral cells AgNP were diluted in DMEM and sonicated for 5 min. Monolayers from the vero cell culture and the AgNP/ DMEM mixture at 1:40 dilution (5×104TCID/ml) were incubated at room temperature with rotation for 1 h. Then, the dengue virus, serotype DEN-2, was added to vero cells, seeded to 90 % confluency. The viral suspension was allowed to absorb for 1 h at 37 °C in 5 % CO2. Following absorption, non-adherent DEN-2 was washed off using phosphate buffered saline (PBS); DMEM supplemented with 2 % FBS was added to the cells. Then, they were incubated at 37 °C in 5 % CO2 for 5 days, which is the time at which the cytopathic effect was observed in about 80 % of the cells infected with

the untreated DEN-2. Each concentration was replicated three times. Virus growth inhibition assay Confluent monolayers of vero cells in 12-well plates were washed with PBS, then infected with DEN-2 at 0.1 multiplicity of infection. The plates were continuously shaken for 45 min at room temperature in AgNP-free conditions, for virus adsorption. The solution was removed and replaced with DMEM medium containing AgNP at 20 μg/ml. Viruses were harvested at 8, 24, 36 h post-infection; the viral yield was estimated by plaque assay on vero cells. Each concentration was replicated five times. As control, DEN-2 infected cells were incubated in AgNP-free medium over different time intervals. A. aegypti rearing Eggs of A. aegypti were provided by the National Centre for Disease Control (NCDC) field station of Mettupalayam (Tamil Nadu, India). Eggs were transferred to laboratory conditions [27±2 °C, 75–85 % R.H., 14:10 (L:D) photoperiod] and placed in 18×13×4-cm plastic containers filled 500 ml of tap water, waiting for hatching. Larvae were fed daily with a mixture of dog biscuits (Pedigree, USA) and hydrolyzed yeast (Sigma-Aldrich, Germany) (3:1, w/w). Larvae and pupae were collected, transferred to glass beakers filled with 500 ml of dechlorinated water, and tested in subsequent experiments (Suresh et al. 2015). Larvicidal and pupicidal toxicity in laboratory conditions Following the method reported by Suresh et al. (2015), 25 A. aegypti larvae (I, II, III, and IV instar) or pupae were placed in a glass beaker filled with 250 ml of de-chlorinated water plus the M. oleifera seed extract (50, 100, 150, 200, 250 ppm) or green-synthesized AgNP (5, 10, 15, 20, 25 ppm). Larval food (0.5 mg) was provided for each tested concentration. Each concentration was replicated five times against all instars. In control treatments, 25 larvae or pupae were transferred in 250 ml of de-chlorinated water. Percentage mortality was calculated as follows: Percentage mortality ¼ ðNumber of dead individuals=Number of treated individualsÞ  100:

Data analysis SPSS software package 16.0 version was used for all analyses. In mosquito experiments, LC50 and LC90 were calculated by probit analysis, following the method by Finney (1971). All

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toxicity data were analyzed using a two-way ANOVA with two factors (i.e., the tested dose and the elapsed time for anti-dengue experiments; the tested dose and the instar for mosquitocidal experiments). Means were separated using Tukey’s HSD test. P