Biocompatible silver nanoparticles reduced from Anethum graveolens leaf extract augments the antileishmanial efficacy of miltefosine

Accepted Manuscript Biocompatible silver nanoparticles reduced from Anethum graveolens leaf extract augments the antileishmanial efficacy of miltefosine Suresh K. Kalangi, A. Dayakar, D. Gangappa, R. Sathyavathi, R.S. Maurya, D. Narayana Rao PII:

S0014-4894(16)30183-7

DOI:

10.1016/j.exppara.2016.09.002

Reference:

YEXPR 7293

To appear in:

Experimental Parasitology

Received Date: 30 September 2015 Revised Date:

3 May 2016

Accepted Date: 9 September 2016

Please cite this article as: Kalangi, S.K., Dayakar, A., Gangappa, D., Sathyavathi, R., Maurya, R.S., Narayana Rao, D., Biocompatible silver nanoparticles reduced from Anethum graveolens leaf extract augments the antileishmanial efficacy of miltefosine, Experimental Parasitology (2016), doi: 10.1016/ j.exppara.2016.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Biocompatible silver nanoparticles reduced from Anethum graveolens leaf extract augments the antileishmanial efficacy of miltefosine Suresh K. Kalangi1Ϯ$, Dayakar. A1Ϯ, Gangappa. D1, R. Sathyavathi2, R. S. Maurya1*, D. Narayana Rao 2*,

Hyderabad – 500 046

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1. Dept. of Animal Biology, School of Life Sciences, University of Hyderabad,

2. School of Physics, University of Hyderabad, Hyderabad- 500046.

Tel: +91-40-23134532, Fax- +91-40- 23010120 2* Corresponding author; e-mail: [email protected] Tel: +91-40-23134335; Fax: +91-40-23010227 Authors have contributed equally

*Combined corresponding authors

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1*Corresponding author; e-mail: [email protected], [email protected]

$ Present Address: Nano Bioscience Laboratory, Institute of Physics, Academia Sinica, Taipei 11529, Taiwan

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Running title: AgNPs augments miltefosine antileishmanial activity

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Abstract Despite the existence of chemotherapy, there is no effective cure for leishmaniasis. In the light of recommended therapeutic regimen is attributed for toxicity and development of

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clinical resistance, exploration of an efficient method of drug delivery could be one of the option in reducing the dosage and toxicity of drugs. This work is aimed in such fashion to study the enhanced antileishmanial activity of miltefosine with silver-nanoparticles (AgNPs) synthesized by using Anethum graveolens (dill) leaf extract as reducing agent. AgNPs were

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synthesized in a single step process and characterized by UV-visible, X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FTIR) to understand the crystal structure and functional groups on their surface. TEM analysis showed that the synthesized AgNPs are

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of an average size of 35nm. By performing MTT assay, we found that, AgNPs (between 20 to 100µM) are biocompatible in nature through pertaining >80% viability of macrophages. Furthermore, AgNPs alone (50µM) have not shown antileishmanial effect on promastigote stage of Leishmania parasite but in combination with miltefosine (12.5µM and 25µM), it magnifies the leishmanicidal effect of miltefosine by ~2-folds (i.e. AgNPs cut down the IC50

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of miltefosine about to half). Scanning electron microscopic (SEM) observation for morphological aberration and genomic DNA fragmentation in promastigotes confirmed the enhanced effect of meltefosine in combination with AgNPs (50µM AgNPs plus 12.5µM miltefosine). Similarly, this combination has likely shown a slight augmentation (p=0.057) of

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miltefosine (2.5µM) leishmanicidal efficacy on amastigote stage of the parasite in infected human macrophages by reducing their intracellular growth.

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Key words: Anethum graveolens; AgNPs; Miltefosine; Leishmaniasis; Nano-carriers and drug delivery. 1

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Abbreviations VL, visceral leishmaniasis; AgNPs, silver nanoparticles; XRD, X-ray diffraction; FTIR, fourier transform infra-red spectroscopy; TEM, transmission electron microscopy; SEM, scanning electron microscopy; IC50, half inhibitory concentration; KBr, potassium bromide; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; MTT, 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DMSO, dimethyl sulfoxide; OD, optical density; UV, ultra violet; AgNO3, silver nitrate; ATCC, American type culture

ACCEPTED MANUSCRIPT 1. Introduction Leishmaniasis is a neglected tropical disease caused by a vector-borne protozoan parasite called Leishmania. It is transmitted by either zoonotic or anthroponotic pattern through phlebotomine sandflies during their blood meal. It is more prevalent and becomes challenging in under developed and developing countries. Among the different clinical forms

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of leishmaniases, visceral infection is most fatal and causes death if remains neglected without treatment. It is commonly known as kala-azar, typically caused by L. donovani in the Indian subcontinent, which primarily infects host macrophages and subsequently invades various lymphoid tissues; eventually causes hepatosplenomegaly. It is estimated that 2-4 lakh

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new cases of visceral leishmaniasis (VL) are being recorded annually around the globe with >10% mortality rate, of which, ~90% only from the Indian subcontinent, Sudan, Ethiopia and Brazil (Alvar et al., 2012). Current therapeutic strategy is relied on antileishmanial drugs

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(Pentavalent antimonials, Amphotercin B, and Miltefosine), in spite of the toxicity, high cost, rapid emergence of resistance, and absence of effective vaccine formulations (Sundar et al., 2007). Apart from this, a rapid co-infection of human immunodeficiency virus (HIV) in VL patients poses a robust challenge in low income countries (Alvar et al., 2008). These circumstances urge for an effective combination therapy involving resistance surpassability

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and safety. Present study is an effort in the view of exploring and in establishing the advantage of biocompatible AgNPs as a synergetic agent or as a drug carrier to augment the efficacy of antileishmanial drug.

For last two decades, nanotechnology has emerged as a rapidly growing field with

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numerous applications in science and technology. The diverse applications in photonics, catalysis, sensing, and medicine (Lin and Wang, 1996; Valden et al., 1998; Gracias et al.,

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2000; Mirkin, 1996; Murphy et al., 2008; Sun and Xia, 2002; Freitas, 2005) have generated a great deal of interest in developing versatile methods to synthesize NPs with well-defined and controlled properties. Unlike, the nanoparticles (NPs) synthesized through chemical routes, with great possibility of toxic effects to use in biomedicine and drug delivery, synthesis based on naturally occurring biomaterials (green synthesis) could be safe due to the natural capping agents on NPs. Typically, plant sources are rich in anti-oxidants and macromolecules like sugars and peptides, which can be exploitable as natural capping agents for specific benefits.

collection; RAW 264.7, mouse macrophage-like leukemic cell line; THP-1, Human monocytic leukemia cell line;

ACCEPTED MANUSCRIPT To explore the NP’s properties made from plant extracts, we followed a single step process of AgNPs synthesis using Coriandrum sativam and Moringa oliefera leaf extracts (Sathyavathi et al., 2010 and 2011). Several other groups also reported on silver/gold NPs using leaf extracts (Begum et al., 2009; Narayanan and Sakthivel, 2008; Philip, 2009; Philip, 2010; Saxena et al., 2012; Shankar et al., 2004) for their biomedical and antimicrobial properties

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(Sharma et al., 2009). In accordance to the previous reports, we have synthesized AgNPs in a single step method for the first time by using dill leaf extract as a reducing agent.

Dill (German word) is an annual herb belongs to the family Apiaceae, genus Anethum, and species A. graveolens. (Plant material which used in present study have been deposited

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at Kakatiya University Herbarium (KUW), Warangal, Telangana State, India with accession no. 1899 for further research purposes.) It is rich in polyphenols, flavonoids, anti-oxidants, essential minerals, and vital vitamins like folic acid, riboflavin, niacin, vitamin A, β-carotene,

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and vitamin C (Jana and Shekhawat, 2010). It has a high medicinal value and been used as an anti-hyperlipidemic agent (Sahib et al., 2012). It has been reported that aqueous extracts of A. graveolens showed a broad-spectrum of anti-bacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Shigella flexneri (Jana and Shekhawat, 2010; Sahib et al., 2012; Arora and Kaur, 2007). In reference of these

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bestowed advantages, in our study, we have tested the dill leaf extract as reducing agent to synthesize the AgNPs with possible biomedical applications, focusing to treat leishmaniasis. 2. Methodology

2.1. Preparation of leaf extract

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The fresh dill leaves (20g) were obtained from fields, thoroughly rinsed with distilled water, and cut into the small pieces. The chopped leaves were boiled in 50ml distilled water

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for 5 min. Subsequently, filtered and cooled to the room temperature, and stored at 4°C till it is used. The filtered extract was used as a reducing and stabilizing agent for 1mM of silver nitrate (AgNO3).

2.2. Synthesis of AgNPs

In a typical one step synthesis of AgNPs, the leaf extract (2.5ml) was added to 50ml of 1mM AgNO3 (99.99%) aqueous solution and incubated at 33°C. The solution was turned from colourless to yellow by 30 min of incubation, indicating the formation of AgNPs. Incubation was further continued until 60 min to facilitate all the reagents are used up in reaction and to form more stable nanoparticles. After synthesis, AgNPs were spun down and washed for twice with excess of distilled water at 10,000 rpm for 30 min, and used in further biological experiments. At regular time intervals (10, 15, 30, 45, and 60 min), synthesized

ACCEPTED MANUSCRIPT AgNPs are collected and used for further characterization. Until unless it is mentioned all experiments in this study are carried out with 60 min AgNPs. 2.3. Characterization of AgNPs The reduced AgNPs solution was collected through periodic sampling and 5ml of aqueous aliquots were used for UV-visible spectrum analysis. In which, the reduction of pure

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Ag+ ions by dill leaf extract was measured at different time intervals (10, 15, 30, 45, and 60 min). Analysis was carried out using a JASCO UV-visible absorption spectrophotometer with a resolution of 1nm between 300 to 800nm. A 300µl sample aliquot was diluted to 10 times with Millipore water to avoid errors due to high optical density of the solution. For X-ray

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diffraction studies, a thin film of the AgNPs was prepared by dipping a glass plate in the solution and the diffraction pattern was recorded by Co–Kα1 radiation with a wavelength of 1.78Å. It was scanned in the region of 20° to 90° for 2θ at 0.02°/min with the 2 sec time

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constant. For FTIR studies, the AgNPs solution was centrifuged at 10,000 rpm for 30 min and the pellet was washed for three times with deionised water to remove uncapping free proteins/enzymes. Then, samples were dried and grinded with potassium bromide (KBr) pellets, and analysed on JASCO FTIR-5300 model, using diffuse reflectance mode, operating at a resolution of 4cm-1. For TEM analysis, the grids were prepared by placing a drop of the

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AgNPs solution on a carbon-coated copper grid and drying under lamp. The structural organization including size and shape of AgNPs were visualized and recorded through the 200kV Ultra High Resolution TEM (JEOL-2010). 2.4. Cytotoxicity of AgNPs on RAW 264.7 cell line

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RAW 264.7 macrophage cell line was obtained from National Center for Cell Science (NCCS), Pune, India. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)

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supplemented with 10% fetal bovine serum (FBS), 200U/ml penicillin, and 200mg/ml of streptomycin at 37°C and 5% CO2 incubator. Culture medium was replaced with fresh medium for every 48h and cells were sub-cultured after trypsinization until reaching confluence. Cell viability was assessed by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide] reduction assay (Mosmann, 1983). Briefly, cells were harvested from 4 to 5 days old culture and seeded in 96-well plate at a density of 5×103 cells/well. AgNPs treatment was given with different concentrations (20, 40, 60, 80 and 100µM) for 48h in a final volume of 100µl/well. At the end of the treatment, 20µl of MTT [5mg/ml prepared in phosphate buffer saline (PBS)] was added and the cells were incubated for next 4h in CO2 incubator. After incubation, 150µl of dimethyl sulfoxide (DMSO) was added and mixed to dissolve the formazan crystals, the optical density (OD) was measured at

ACCEPTED MANUSCRIPT 540nm using a microplate reader (Quant Bio-tek Instruments, Inc.), and the percentage of cell viability vs AgNPs concentration (µM) was drafted. 2.5. Culturing of Leishmania parasite Promastigotes of L. donovani Dd8 strain obtained from ATCC (American type culture collection, U.S.A) and cultured in M199 medium supplemented with 15% heat-inactivated

streptomycin at 25°C±1. 2.6. Cytotoxicity of AgNPs on promastigotes

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FBS, 20mM HEPES, 4mM NaHCO3, pH7.4, 100U/ml of penicillin and 100mg/ml of

Cytotoxicity assay was performed by MTT assay as mentioned in section 2.4.

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Exponentially growing log phase promastigotes (1×106 cells/well) were seeded in a 96-well microtiter plate and treated with 50µM AgNPs alone or in combination of 12.5µM and 25µM miltefosine; which would be IC25 and IC50 on L. donovani (MHOM/80/IN/Dd8)

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promastigotes respectively (Verma et al., 2004). Miltefosine alone treated parasites were used as positive control. The percentage of promastigote viability (%) vs AgNPs (50µM) alone and in combination of miltefosine was drafted.

2.7. Promastigotes morphological analysis by SEM

For SEM analysis, samples were prepared as described earlier (Li et al., 2009 and

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Dayakar et al., 2012). Briefly, promastigotes of both untreated (cont rol) and t reated with 50µM AgNPs alone and in combination with 12.5µM miltefosine were fixed for 3h with 2.5% (v/v) glutaraldehyde prepared in 1X PBS (pH7.4) at 4°C, which followed by post fixation with 1% (w/v) osmium tetroxide (OsO4) for 30 min. After fixation step, they we r e

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processed in ethanol series viz. 30%, 50%, 70%, 90%, and three times in 100%. Finally, samples were subjected for point drying using liquid CO2 and coated gold particles. The

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images were captured by using XL30 ESEM make FEI. 2.8. DNA fragmentation assay Genomic DNA was extracted from miltefosine alone, AgNPs (50µM) alone, and AgNPs plus 12.5µM miltefosine treated promastigotes after 48h using standard protocol. Briefly, promastigotes were obtained from culture by centrifugation at 4000g for 10 min and resuspended in lysis buffer (10mM Tris, 10mM ethylenediaminetetraacetate (EDTA), 0.5% w/v sodium-N-laurylsarcosine pH7.5) and added proteinase-K (100µg/ml), allowed to digest by overnight incubation at 50°C. Next day, RNaseA (0.3mg/ml) was added and incubated for 1h at 37°C, then, lysates were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) at 16,000g for 5 min. Further, supernatant was treated with 0.1 volume of 3M sodium acetate (pH5.5) and 2 volumes of ethanol, incubated for overnight at 20°C. Next day, it was spun

ACCEPTED MANUSCRIPT down at 16,000g for 10 min and then DNA was washed with 95% ethanol. Further, it was subjected for air drying and dissolved in 10mM Tris-1mM EDTA buffer (pH8.0), subsequently quantified by using spectrophotometer at 260/280nm. It was allowed to run on a 1% agarose gel for 1h by supplying 100V current and the fragmentation patter was visualized under UV light, and snapped the picture using Gel doc 2000 (Bio-Rad).

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2.9. Light microscopic analysis of infection rate in human macrophage cell line Human monocytic leukemia cell line (THP-1) was seeded into the 8-well chamber slides and stimulated with 10ng/ml phorbol 12-myristate 13-acetate (PMA) by incubating at 37oC and 5% CO2 for 24h to transform into a macrophage like phenotype. Thereafter,

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macrophages (2×104) were infected with early stationary phase promastigotes for 6h by adding 10:1 ratio of parasite to macrophage. Non-internalized parasites were removed by washing with sterile 1x PBS and infected cells were treated with 50µM AgNPs alone or in

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combination with miltefosine (2.5µM) for 48h. After completion of treatment, slides were stained with Giemsa stain and infection rate (amastigotes number per 100 macrophages) was counted by using light microscopy (Dayakar et al., 2015). 2.9. Statistical analysis

Data was analysed using two-tailed unpaired t-test and significance with p≤0.05 and

3. Results

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p≤0.01 was represented as * and ** respectively.

Single step synthesis of AgNPs was successfully achieved by using dill leaf extract. As shown in the fig.1b (tube A: 1mM AgNO3, tube B: dill leaf extract, and tube C: reduced

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AgNPs 60 min), AgNPs were shown to be reduced with dill leaf extract after 30 min of reaction and turned into yellow colour, indicating the formation of AgNPs thereafter. Later

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the same was supported by further characterization of synthesized NPs. 3.1. UV-visible spectra inferring the completion of AgNPs synthesis Despite the visible colour change was indicating the successful synthesis of AgNPs; it was further analyzed by UV-visible absorption spectroscopy. In which, the progress of the reaction between metal ions (Ag+) and leaf extract was monitored at different time points (10, 15, 30, 45, and 60min). Results demonstrated that there was no significant peak signature for 10 and 15 min reaction times, indicating that no synthesis of AgNPs yet. But after 30 min, as the solution was turning into yellow colour, we could able to see an increased absorption of the solution in visible range of UV-spectra (fig.2). 3.2. XRD spectra indicating crystal structure of AgNPs

ACCEPTED MANUSCRIPT A typical XRD pattern of AgNPs as shown in fig.3, the diffraction peaks at 37.79, 44.84 and 77.53° were correspond to the (111), (200), and (311) facets of the face centred cubic crystal structure. The elevation of the Bragg peaks indicating the formation of AgNPs. The unassigned peaks, marked with * in the fig.3 could be due to the crystallization of bioorganic phase that occurs on the surface of the AgNPs.

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3.3. FTIR spectra depicting the presence of functional groups on AgNPs It was carried out to identify the potential biomolecules in the leaf extract such as peptides for AgNPs reduction and capping reagent for the stability of reduced AgNPs (Mosmann, 1983; Sahib et al., 2012; Arora and Kaur, 2007; Kulikova et al., 2015). A typical FTIR spectrum of the synthesized AgNPs (fig.4b) and dill leaf extract was shown in (fig.4a)

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figure 4. The strong absorption peaks identified at 3,309 cm−1 resulting from the stretching of -NH- band (amino groups) or -OH- band (hydroxyl group) due to phenols in the leaf extract.

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The absorption peaks at about 2916 cm−1 could be assigned to stretching vibrations of C-H alkanes and an intense band at 1599 cm−1 is the characteristic of amino acids containing NH2- groups. The peaks at 1406 and 1243 cm−1 could be assigned to the C-H deformation, ketones and C-O stretch respectively, and an intense band identified at 1022 cm−1 is due to PO-stretch (fig.4a). The reduced AgNPs have shown strong absorption peaks at 676, 1523,

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1675, 2348, and 3753 cm-1; an intense band at 676 cm-1 could be assigned to -CH- out of plane bending vibrations are substituted ethylene systems -CH=CH- (cis). A strong peak at 2348 cm-1 could be assigned to the silver metal linked -CN- bond from organic sources (Miller and Wilkins, 1952) indicates the presence of NPs in the sample and a peak at 1675

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cm-1 represents the C=O stretch of amides of proteins or peptides formed as capping agent derived from dill leaf extract (Kulikova et al., 2015). Finally, the presence of a strong peak at

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3753 cm-1 could be assigned to the primary amines, an evidence for the presence of peptides on AgNPs (fig.4b).

3.4. TEM analysis indicating structural organization of AgNPs Typical TEM images of synthesized AgNPs at different reaction time points were shown in fig.5a-5d. At an early time point (30 min), the obtained AgNPs were mostly irregular, agglomerated, and larger in size (fig.5a), after 45 min, the isolated AgNPs were still irregular in shape, indicating reaction was not yet completed (fig.5b). AgNPs isolated at 60 min were well organised, spherical, and an average size about 35nm (between 8-100nm) (fig.5c). The fig.5d shows the histogram taken from large number of micrographs and fig.5e represents a selected area electron diffraction (SAED) form a cluster of AgNPs.

ACCEPTED MANUSCRIPT 3.5. AgNPs showing biocompatibility to the macrophages AgNPs (60 min) have shown only ≤18% of cytotoxicity on RAW 264.7 mouse bloodderived macrophage cell line at maximum concentration (100µM) and ~90% of viability was observed at 50µM AgNPs (fig.6). The other AgNPs synthesized at 30 and 45 min were also tested but had no effect (data not shown). Results suggest that the dill leaf extract usage have

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been advantageous in synthesizing biocompatible AgNPs, it might be due to the presence of peptides or amino acids as capping agents, while reducing the AgNPs. Unlike the chemically synthesized AgNPs, which are supposed to address many toxicity related issues, these AgNPs would not cause any toxicity and immune defects. It would be a potential nanocarrier for

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effective drug delivery in leishmaniasis chemotherapy.

3.6. AgNPs reducing miltefosine inhibitory concentration against promastigotes AgNPs of different concentrations (100, 50, 25, 12.5, 6.125 and 3.062µM) were tested

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against promastigotes, but none of them has shown antileishmanial activity (data not shown). Therefore, we randomly selected 50µM AgNPs concentration for combinational study with miltefosine. Interestingly, in promastigotes, AgNPs at 50 µM in combination with meltefosine at two different testing concentrations i.e. 12.5 and 25 µM have induced ~21% and ~33% more death respectively than meltefosine alone in a dose dependent manner. It suggests that

concentrations (Fig.7).

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AgNPs significantly (p≤0.01) augments the antileishmanial activity of miltefosine at lower

3.7. AgNPs with the combination of miltefosine inducing morphological aberrations in promastigotes

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SEM captured images clearly suggesting the loss of structural integrity in treated promastigotes with miltefosine (12.5µM) in combination of AgNPs (50µM). Parasites lost

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their flagellum and shape, become ovoid, appearing as like apoptotic cells. On the other hand, control promastigotes have their intact morphology with spindle shaped body and anteriorly postulated flagellum (fig.8). It suggests that, with the combination of miltefosine, AgNPs could induce apoptosis-like death as shown by miltefosine alone (Paris et al., 2004). 3.8. AgNPs with the combination of miltefosine creating DNA breaks in promastigotes Genomic DNA fragmentation by endogenous nucleases is the hallmark of apoptosis, is a late remarkable event. Here, the AgNPs (50µM) in combination with miltefosine (12.5µM) induces DNA breaks in L. donovani promastigotes (fig.9), suggesting their apoptosis-like death, as observed with miltefosine alone (Paris et al., 2004). 3.9. AgNPs with the combination of miltefosine reducing infection rate in macrophages

ACCEPTED MANUSCRIPT Microscopic results claimed that the amastigotes number was significantly reduced in miltefosine alone (p≤0.05) and in combination with AgNPs (p≤0.01) treated macrophages, but such an effect was not significant with AgNPs alone (fig.10B). It seems, AgNPs slightly enhances (p=0.057) miltefosine antileishmanial activity on intracellular amastigotes.

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Microscopic images of infection in the macrophages were depicted in fig.10A.

4. Discussion

Prevention of leishmaniasis is remarkably relied on chemotherapy. The well known antileishmanial drugs are pentavalent antimonials, pentamidine, amphotericin B, miltefosine,

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paromomycin, allopurinol, and many other drugs at various stages of their developmental process (Loiseau and Bories, 2006; Croft et al., 2006). Among all, miltefosine is thought be the first effective and safe oral drug; hence it has been licensed for 10 years in India for the

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treatment of visceral leishmaniasis. But later it proved to be having gastrointestinal side effects, and teratogenic potentiality (Dorlo et al., 2012). Apart from the health complications coupled with chemotherapy, drug resistance in endemic regions is another major concern on a global scale. It is emerging due to the free accessibility of drugs and their widespread misuse (inappropriate dosages) in rural areas (Sundar et al., 1994). Apart from this, the mechanism of

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drug resistance includes; decreasing intracellular drug pressure either due to decreased uptake or increased efflux or sequestration of active drug by thiole conjugation. It typically handled by the amplified expression of P-glycoprotein A (P-gpA), a multi-drug resistance protein transporter (Perez-Victoria et al., 2002) or it can be independent of P-gpA, as observed with

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miltefosine (Croft et al., 2006). In response to this, researchers have focused on searching for the resistance striking agents (Perez-Victoria et al., 2002). In order to this, it is interesting to

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develop strategies for increasing intracellular drug accumulation by means of efficient drug entry or delivery that brings down the dosage and its allied toxicity. In these circumstances, biosynthetic AgNPs combination would be much better alternative medicine for prescribing to treat leishmaniasis and its co-infections. In this study, the biosynthetic AgNPs combination is shown to enhance leishmanicidal effect of miltefosine. In which, AgNPs synergistically cut down the dosage of miltefosine to about half on promastigote stage of the parasite. Unlike the other synergetic studies; amiodarone or amphotericin B with the combination of miltefosine (Ménez et al., 2006; Serrano-Martín et al., 2009) or chemically synthesized NPs (Handy and Shaw, 2007), which produce antagonism and common side effects, the reduced AgNPs with dill leaf extract used in this study have endorsed for no considerable toxicity on host cells and attained a potent

ACCEPTED MANUSCRIPT synergetic effect with miltefosine by amplifying its action against Leishmania parasites. This study may help to explore the ways for increasing miltefosine intracellular retention to abort the parasite persistence or its drug resistance. During AgNPs synthesis using dill leaf extract, the colour change in reaction mixture was started at 30 min, indicating the starting point of the AgNPs reduction and it continued till

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60 min, is authenticated by UV-spectra. It results in the formation of AgNPs with different shapes and sizes predicted by TEM. Most spherical and structurally organized AgNPs used in this study were obtained at 60 min time point. Other reaction times include irregular shaped and aggregated AgNPs, indicating incomplete reduction of AgNPs with dill leaf extract. This

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can be plausibly explained by the amount and length of the peptides on NPs that could directly influence the stability and size of NPs. It would be most appropriate reason for obtaining various sizes as peptides get stabilizing on NPs at different reaction times (Ansary,

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2013).

Absence of phenol corresponding peek (refers to -OH broad peak) at 3,309 cm-1 (fig. 4a) and presence of primary amine corresponding peak at 3753 cm-1 illuminating the involvement of carboxyl (−OH) and amine (N-H) groups of dill leaf extract in reduction of Ag+ ions to Ag0 NPs. The FTIR spectroscopic study also confirmed that the protein present in

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the extract acting as a stabilizer for the AgNPs and preventing the aggregation of particles. Biosynthetic AgNPs showed no cytotoxic effect as represented in fig.6 at different dosages on macrophage cells. Its biocompatibility would be mostly reflected by the presence of natural compounds as reported previously using other plants (Sharma et al., 2009, Prabhu and

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Poulose, 2012). Surprisingly, none of the AgNPs (30, 45 and 60 min) alone could induce death in Leishmania promastigotes (data not shown) but in combination with miltefosine, 60

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min AgNPs at 50µM concentration could intensify the leishmanicidal effect of miltefosine. Entry and enhancive leishmanicidal effect of particles can be explained by variation in their size and charge, with and without combination of miltefosine. In the present study, 60 min AgNPs are mostly spherical in shape, which might have helped them to enter into the cells. It is clear that Ag+ ions reduced to Ag0 by the peptides having NH2, -COOH, and -OH groups, so the total charge is neutral, which might have restricted them to enter into the cells or the entered NPs might not be sufficiently enough to show effect. Though, peptides from dill leaves possess different properties, as capping agents, they may have obscured the AgNPs anti-parasitic activity (Kulikova et al., 2015). Therefore, no antileishmanial effect was observed with AgNPs alone. On the other hand, when they mixed with miltefosine, it is likely to have weak interactions; like hydrogen bonds between one of the carboxyl or hydroxyl

ACCEPTED MANUSCRIPT groups of peptides present on AgNPs with NH2 / P-O4, which alters the total net charge and facilitates their entry into the cells. Probably, these interactions could aid for the accumulation of more drug molecules in promastigotes. Moreover, miltefosine is an alkylphosphocholine drug with long fatty acid like chain; it might be an added advantage to NPs for crossing the plasma membrane and also for effective functioning, as in chemotherapy of cancer with

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conjugation of long chain fatty acid composed molecules (Dorlo et al., 2012). Thus AgNPs could facilitate miltefosine to be effective even at lower concentrations. These results are duly supported by previous reports on various NPs entry into the cells based on their charge variations or capping agents (Verma et al., 2008; Verma and Stellacci, 2010) and tracing of 5-

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flurouracil conjugated quantum dots (Kalangi et al., 2012) in breast cancer cells. We believe one of the proposed ways might be plausible justification for an enhancive effect of AgNPs on miltefosine activity. SEM images for revealing loss of structural integrity in promastigotes

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and DNA fragmentation studies further supported our study in proving that apoptosis-like death induced by the combination of miltefosine and AgNPs. Its leishmanicidal effect on amastigotes in macrophages is partially contributing for aforementioned justification about their interaction and entry into the cells. However, further studies are needed to explore the actual synergetic mechanism for enhancive miltefosine leishmanicidal effect on both stages of

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L. donovani parasite to implicate these NPs as perfect nano-drug carriers. Conclusion

To the best of our knowledge this study claims the first evidence in cut down the IC50 of miltefosine by using biocompatible NPs in combination, against Leishmania parasites. It

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creates a new hope for better chemotherapy against leishmaniasis. These AgNPs can be effective as special category nano-drug carriers with no effect either on the parasites or host

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cells but thought to be involving in increased availability of drug via unknown mechanism. This study also reports a simple, cost-effective, and green biosynthesis of AgNPs using dill leaf extract for the first time and their use in biomedical applications. Acknowledgments

We greatly acknowledge financial support from the DST-PURSE fellowship from University of Hyderabad provided for this work. Conflict of interest: Authors have declared no conflict of interest

References

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Photographs showing (a) dill leaf and (b) tube-A: 1mM AgNO3, tube-B: leaf extract, and tube-C: reduced AgNPs at 30 min of reaction appearing in yellow or golden amber colour. Figure 2. UV-visible spectra recorded as a function of time of the reaction of an aqueous

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solution of 10-3M AgNO3 with A. graveolens leaf extract (a) 10 min (red) (b) 15 min (black) (c) 30 min (gray) (d) 45 min (blue), and (e) 60 min (pink). After 30 min, it is showing a significant peak signature, which represents initiating the formation of AgNPs in the solution and an increased absorption in visible range of the spectrum. Y-axis represents absorbance of

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the solution and X-axis represents wavelength of UV-visible light.

Figure 3. Picture depicts a typical XRD pattern of AgNPs synthesized by dill leaf extract. Here, the diffraction peaks observed at 37.79, 44.84 and 77.53° corresponding to the (111),

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(200), and (311) facets of the face centred cubic crystal structure. Bragg peaks elevation indicates the formation of AgNPs and unassigned peaks marked with * might be due to the crystallization of bioorganic phase occurs on the surface of the AgNPs. X-axis represents 2ɵ (degrees) and Y-axis represents the peak intensity.

Figure 4. FTIR spectrums of A. graveolens leaf extract (a) and reduced AgNPs (b). X-axis represents the wavelength (cm-1) and Y-axis represents the transmission. (a) The strong

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absorption peaks are identified at 3,309 cm−1 (stretching of -NH- or -OH band of phenols), 2916 cm−1 (stretching vibrations of C-H alkanes), 1599 cm−1 (characteristic of NH2 group of amino acids), 1406 cm−1 (C-H deformation), 1243 cm−1 (ketones and C-O stretch), and 1022

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cm−1 (P-O stretch) might be due to the concerned events. (b) The strong absorption peaks are noticed at 676 cm-1 (-CH- group out of plane and bending vibrations are substituted ethylene systems -CH=CH), 2348 cm-1 (silver metal linked to the -CN- bond from organic sources),

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and 1675 cm-1 (C=O stretch of amides of proteins or peptides) might be due to the concerned events eventually leads to the AgNPs synthesis. Figure 5. TEM images showing irregular, agglomerated, and larger AgNPs at (a) 30 min & (b) 45 min, and (c) well organised, spherical AgNPs at 60 min. (d) Histogram of 60 min AgNPs showing particle size (nm) vs counts and reveals the average size of AgNPs as ~35nm. (e) Selected area electron diffraction patteren of AgNPs cluters. Figure 6. Picture depicts the biocompatability of dil leaf extract reduced AgNPs on RAW 264.7 mouse macrophage cell line. X-axis represents the concentration (20, 40, 60, 80, and 100 µM) of AgNPs (60 min) and Y-axis represents the percentage of cell viability. All the different concentration of AgNPs are showing >80% macrophage viability.

ACCEPTED MANUSCRIPT Figure 7. Picture depicts the enhancive antileishmanial effect of miltefosine in combination with AgNPs. X-axis showing different experimental variables and Y-axis showing percentage of promastigote viability. Here, the AgNPs (50µM) in combination with 12.5µM and 25µM miltefosine (Milt) showing ~21% and ~33% additional death (p≤0.01) in promastigotes respectively compared to its respective miltefosine doses alone induced death. But AgNPs

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(50µM) alone are ineffective against promastigotes and untreated promastigotes served as a control in the study.

Figure 8. SEM pictures depict the morphological changes in promastigotes. (b) AgNPs (50µM) in combination with 12.5µM miltefosine showing loss of structural integrity in

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promastigotes compared to (a) untreated promastigotes and (c) AgNPs (50µM) alone treated promostigotes appeared as untreated promastigtes without loss in structural intgrity. Figure 9. Picture depicts the genomic DNA fragmentation pattern of L. donovani

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promastigotes on 1% agarose gel. Lane 1: DNA ladder, Lane 2: Miltefosine (12.5µM) treated promastigotes DNA, Lane 3: AgNPs (50µM) plus 12.5µM miltefosine treated promastigotes DNA, and Lane 4: AgNPs (50µM) alone treated promastigotes DNA. Here, miltefosine alone and in combination with AgNPs showing definite fragmentation (lane 2 & 3 respectively), unlike AgNPs alone, which is showing no DNA breaks in the parasite genome (lane 4).

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Figure 10.A. Light microscopic picture depicts (A) the intracellular amastigotes in macrophages stained with Giemsa stain and (B) the rate of infection. In the fig.10A, macrophages (a) Uninfected (b) Infected and untreated (c) Infected and AgNPs (50µM) alone treated (d) Infected and treated with 50µM AgNPs plus 2.5µM mitefosine. In the fig.10.B, X-

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axis represents the different experimental variables and Y-axis represents the number of intracellular amastigotes per 100 macrophages. Here, AgNPs alone are ineffective (ns; non-

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significant), while in combination with 2.5µM mitefosine, it is showing significant (p≤0.01) reduction in amastigotes count against infected and untreated macrophages (control). Moreover, miltefosine alone treated macrophages also showing significant (p≤0.05) reduction in infection rate compared to control, but it is one * less significant than that of combination. Here, the AgNPs shown to augment the miltefosine leishmanicidal effect on amastigote stage of the parasite by p=0.057.

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Highlights AgNPs reduced from Anethum graveolens (dill) leaf extract

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AgNPs characterized by UV-visible, XRD, FTIR and TEM.

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AgNPs, biocompatible in nature to macrophages and also has no antileishmanial

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activity

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But in combination, AgNPs augments the miltefosine antileishmanial activity i.e. IC50

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shown to be reduced.

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•