Ethics statement

The study was approved by and carried out under the guidelines of the Ethical Committee of the Indian Institute of Chemical Biology, Kolkata. All subjects who participated in this study provided informed consent in writing according to the Indian Institute of Chemical Biology guidelines and approval. The animal experiments were approved by the Animal Ethical Committee (147/1999/CPSCEA) of the institute, according to the National Regulatory Guidelines issued by the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), under the Division of Animal Welfare, Ministry of Environment and Forest, Government of India.

Animals and parasite

BALB/c mice, bred in the animal house facility of Indian Institute of Chemical Biology (Calcutta, India), were used for the experiments. For parasite maintenance the L. donovani strain AG83 (MHOM/IN/1983/AG83), originally isolated from an Indian kala-azar patient was periodically injected in appropriate numbers in 4–6 weeks old Syrian golden hamsters (Mesocricetus auratus) reared in pathogen-free animal care facility of the Indian Institute of Chemical Biology. The infected hamsters were sacrificed at two months infection to isolate L. donovani amastigotes from the spleens. These amastigotes were transformed into promastigotes in M199 supplemented with 10% FCS, 2 mM glutamine, penicillin G (100 U/ml) and streptomycin sulfate (100 ug/ml) at 22°C.

Reagents

RPMI 1640, M199, penicillin, streptomycin, PEG-Catalase (PEG-Cat) and PEG-Superoxide dismutase (PEG-SOD) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Fetal calf serum (FCS) was from (GIBCO/BRL, Grand Island, NY). N-Acetyl-L-cysteine (NAC) and NG-monomethyl L-arginine (L-NMMA) was purchased from Merck (GmBH, Germany). MitoTracker deep red and MitoSOX^™ red were obtained from Molecular Probes (Eugene, OR, USA). The new formulation of liposomal amphotericin B, Kalsome^™10, was a kind gift from Lifecare Innovations Pvt. Ltd. Gurgaon, India. Conventional liposomal formulation of AmB (AmBisome) was purchased from Gilead Science, Inc., (USA). Amphotericin B deoxycholate was purchased from Bharat Serums and Vaccines Limited (India).

Formulation of KalsomeTM10 and Ambisome

KalsomeTM10 is a sterol enriched, mixed lamellarity, amphotericin B intercalating liposome in 0.9% saline, wherein, the concentration of amphotericin B is 0.2 mg/mg total lipid. Ergosterol constitutes almost 50% of the total liposomal lipid, overall consisting of phospatidylcholine, ergosterol and AmB in 5:2:1.8 molar ratios [8].

Ambisome is unilamellar bilayer liposome with amphotericin B intercalated within the membrane wherein, the concentration of amphotericin B is 0.143 mg/mg total lipid [11].

Nanoparticle Tracking Analysis (NTA)

NTA measurements were done with a NanoSight LM20 (NanoSight, Amesbury, United Kingdom), according to the recommended protocol using appropriate dilutions of KalsomeTM10 and Ambisome. The software used for capturing and analyzing the data was the NTA 2.0 Build 127. The mean size and SD values were obtained using the NTA software. These values correspond to the arithmetic values calculated with the sizes of all the particles analyzed by the software.

Cell viability assay

AlamarBlue^® reagent (Life Technologies, Carlsbad, USA) was employed for L. donovani promastigote and mouse peritoneal macrophage cell viability assay. Live cells maintain a reducing environment within their cytosol. AlamarBlue^® reagent contain Resazurin/AlamarBlueH (7-Hydroxy-3H-phenoxazin-3- one 10-oxide) as an active ingredient which is non-toxic and cell permeable. This active compound is blue in colour and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin which is red in colour and highly fluorescent. Hence, viable cells continuously convert resazurin to resorufin thereby increasing the overall fluorescence and colour of the media surrounding the cells. Assays were performed in sterile 96-well plates using 100 μl of late log-phase promastigotes adjusted to (1x10⁷ cells/ml) and 5x10⁵ macrophages in 100 μl of medium. These cells were incubated in the absence (control) or presence of varying concentrations of KalsomeTM10, Ambisome and amphotericin B for different time periods. After the completion of treatment, 10 μl of the resazurin dye (0.01%) was added to these cells, and were incubated for a further 4 h at 37°C. We have seen that culturing the promastigotes at 37°C for 4 h does not affect the viability of the parasites as also shown by an earlier report [12]. Cells were then analyzed in a MicroQuant microplate reader (Biotek-Instrument Inc., Winooski, VT, USA) at a wavelength of 570 nm, using 600 nm as reference wavelength (normalized to the 600 nm value). Cell viability was evaluated based on comparison with untreated control cells. ED₅₀ and ED₉₀ defined as the KalsomeTM10 concentrations that inhibited resazurin reduction with respect to control parasites by 50% and 90%, respectively, were calculated using sigmoidal regression analysis (Microsoft Excel).

Determination of cellular morphology in L. donovani promastigotes

L. donovani promastigotes (1×10⁷ cells/ml) were treated with different concentrations of KalsomeTM10, and incubated at 22±2°C for 4 h. The cells were then harvested, resuspended in PBS (pH 7.4), and observed under optical microscope (Laika DMI 4000, Leica and Zeiss Co., Cambridge, England). The cellular morphology was recorded through QWin software and images were processed using Adobe Photoshop 5.5 (Adobe Systems, Inc., Mountain View, CA, USA) software. For scanning electron microscopy KalsomeTM10 treated and untreated cells were first quick-fixed in 2.5% glutaraldehyde and then post-fixed with 1% OsO4. These processed cells were gradually dehydrated and air-dried. They were finally sputter-coated with gold palladium and examined in a JEOL JSM-5200 electron microscope using an accelerating voltage of 20 kV.

Evaluation of Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) binding of cells through flow cytometry

Binding of Annexin V-FITC/PI to cells was assessed using FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen^™, New Jersey, USA) according to the manufacturer’s instructions. Briefly, 100 μl of promastigotes (1x10⁷/ml) incubated with varying concentrations of KalsomeTM10, Ambisome and amphotericin B for different time periods were harvested and washed twice with cold PBS. Then they were resuspended in 100 μl binding buffer and transferred to a 5 ml culture tube. 5 μl of FITC Annexin V was added and the cells were incubated for 15 min at room temperature (25°C) in the dark. 400 μl of binding buffer was further added to each tube along with 5 μl PI. The cells were then analysed on a flow cytometer (Becton Dickinson, LSR Fortessa, New Jersey, USA) using FACS Diva software (BD Biosciences, New Jersey, USA).

Measurement of mitochondrial membrane potential (Δѱ_m) and mitochondrial integrity

Mitochondrial membrane potential (Δѱ_m) was measured using the BD^™ MitoScreen flow cytometry mitochondrial membrane potential detection kit, according to the manufacturer’s instructions. Briefly, 100μl of KalsomeTM10-treated and non treated L. donovani promastigotes (1x10⁷/ml) were collected and centrifuged at 400 × g for 5 minutes at room temperature. Supernatant was then carefully removed and the cells were resuspended in 0.5 ml of freshly prepared working solution of JC-1 dye and incubated for 10–15 min at 37°C in a CO₂ incubator. Subsequently, the stained cells were washed twice and resuspended in 0.5 ml of assay buffer for analysis by flow cytometry in Becton Dickinson, LSR Fortessa using FACS Diva software.

Evaluation of intracellular ATP depletion

The depletion of the intracellular ATP pool in KalsomeTM10 treated and untreated cells was measured using the ATP determination Kit (Molecular Probes) according to the manufacturer's instructions. Briefly, the cells were washed and then resuspended in PBS. To this cell suspension, 9 volumes of boiling 100 mM Tris, 4 mM EDTA pH 7.75 was added and further incubated for 2 min at 100°C. Cells were then centrifuged and the supernatants transferred to a fresh tube on ice until measurement. The sample lysate was then mixed with the reaction buffer provided in the kit and the concentration of ATP was determined using ATP standard curve generated from the readings on Synergy H1 Hybrid reader (BioTek).

Determination of intracellular reactive oxygen species (ROS) generation

Intracellular H₂O₂ levels in treated and untreated cells were measured using the ROS-Glo assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The KalsomeTM10 treated and untreated cells were added to an opaque white 96 well plate in RPMI media. The H₂O₂ substrate solution was then added, making the final volume to 100 μl. The plate was then incubated at 37°C in a 5% CO₂ incubator for 60 min. 100 μl of the ROS-Glo detection solution was then added to each well. After additional 20 min incubation at room temperature, bioluminescent readings were obtained using a Synergy H1 Hybrid reader (BioTek). Mitochondrial superoxide in KalsomeTM10 treated and untreated cells was measured using MitoSOX^™ red and analysed on a flow cytometer (Becton Dickinson, LSR Fortessa) with FACS Diva software.

Determination of intracellular glutathione (GSH) content and lipid peroxidation products

Intracellular GSH content in treated and untreated cells was measured using the Glutathione Fluorometric Assay Kit (Biovision, USA) according to the manufacturer's instructions. Briefly, the sample lysate was mixed with the reaction buffer provided in the kit and the concentration of GSH was calculated from the GSH standard curve generated using the readings on Synergy H1 Hybrid reader (BioTek). For the estimation of lipid peroxidation products, the treated and untreated cells were washed twice with PBS and suspended in 1 ml of 15% sodium dodecyl sulfate (SDS)-PBS solution. The fluorescence intensities of the total fluorescent lipid peroxidation products was then obtained [13] through readings on Synergy H1 Hybrid reader (BioTek) with excitation at 360 nm and emission at 430 nm.

Evaluation of in vivo caspase-like activity

In vivo caspase-like activity in KalsomeTM10, Ambisome and amphotericin B treated and untreated cells were determined using Intracellular Caspase Detection ApoStat kit (R&D Systems, Inc., Minneapolis, USA) according to the manufacturer's instructions. Briefly, cells were stained directly during the last 30 minutes of the apoptosis induction period. For this, 1 μl of ApoStat per 100 μl of culture volume was added. The staining was performed at 37°C. Cells were then harvesetd into 1 ml tubes, centrifuged at 500 x g for 5 minutes and washed once with 1 ml of PBS to remove unbound reagent. The cells were then resuspended in 500 μl of PBS for analysis on a flow cytometer (Becton Dickinson, LSR Fortessa) with FACS Diva software.

Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay

TUNEL assay was performed according to the manufacturer’s instructions (ApoAlert^™ DNA Fragmentation Assay Kit, Takara Bio, Inc., USA). Briefly, 500 μl of KalsomeTM10 treated and untreated promastigotes was harvested by centrifugation and washed twice with 1 ml of PBS. The cells were then resuspended in 0.5 ml of PBS and fixed by adding 5 ml of fresh, prechilled 1% formaldehyde/PBS and incubated at 4°C for 20 min (step repeated two times). Resuspended cells in 0.5 ml of PBS were then permeabilized by adding 5 ml of 70% ice-cold ethanol and incubated at –20°C for at least 4 h. The cells were washed twice in PBS and then transferred to an amber 1.5-ml microcentrifuge tube (to protect samples from light). The cells were then resuspended in 80 μl of equilibration buffer and incubated at room temperature for 5 min. The cells were washed with PBS and resuspended in 50 μl of TdT incubation buffer and incubated at 37°C in a water bath for 60 min, protected from direct light. Then 1 ml of 20 mM EDTA was added to terminate the reaction, and mixed by gentle vortexing. The cells were pelleted down by centrifugation and resuspended in 1 ml of 0.1% Triton X-100/BSA/PBS. The cells were then resuspended gently in 0.5 ml of PI/RNase/ PBS to stain with PI following incubation at room temperature in the dark for 15–30 min. They were analyzed on a flow cytometer, Becton Dickson Canto II with BD FACS Scan Software (San Diego, CA, USA).

DNA laddering experiment

Fragmentation of DNA into oligonucleosomal bands, as a function of apoptotic cell death, was studied by DNA laddering assay as described before [14]. Apoptotic DNAs from 1 ml of KalsomeTM10 treated and untreated L. donovani promastigotes (1x10⁷/ml)) were isolated using Suicide Track^™ DNA Ladder Isolation Kit (Calbiochem, Germany). Briefly, the treated and untreated cells were suspended in 500 μl of extraction buffer which separates apoptotic DNA from high molecular weight DNA. The cells were then incubated on ice for 30 min and centrifuged at 15000 x g for 5 min. Supernatants carefully removed and transferred to a clean tube. RNA in the supernatant was then degraded using solution 2 of the kit. Subsequently, DNA from the cell lysate was isolated using solution 3 and incubation at 50°C for 1 h. The DNA pellet obtained after centrifugation was washed with 70% ethanol followed by 100% ethanol. It was further air dried and resuspended in buffer (10 mM Tris pH 7.5, 1 mM EDTA). DNA aliquots were electrophoresed on 1.5% agarose gel containing ethidium bromide (0.5μg/ml), using Tris—acetate—EDTA (pH 8.0) running buffer, and then observed and photographed under UV illumination in a BioRad gel documentation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell cycle analysis

Cell cycle analysis of KalsomeTM10 treated and untreated L.donovani promastigotes was done using flow cytometry. After treatment of promastigotes with varying concentrations of KalsomeTM10 for different time periods, the cells were harvested and washed thrice with PBS and fixed in 50% ethanol (diluted in PBS) for 4 h. The fixed cells were then washed thoroughly, treated with 100 μg/ml RNase A and suspended in 1ml of staining solution. After 1 h, the cells were stained with 40 μg/ml PI for 20 min at room temperature. The percentage of cells in G1, S and G2/M phases of the cell cycle were determined in Becton Dickson Canto II flow cytometer and with BD FACS Scan Software.

Isolation of peritoneal macrophages and in vitro evaluation against intracellular amastigotes

BALB/c mice, 6–8 weeks old were used for the experiments. Mouse peritoneal macrophages were collected by infusing the peritoneal cavity with ice-cold sterile RPMI supplemented with 3% FCS. For in vitro infection studies 5 x 10⁶ peritoneal macrophages were cultured on cover slips and incubated overnight. Next day unattached cells were washed of and then infected with parasites at 1:10 ratio. After 3 h of incubation the unbound parasites were removed by washing thrice with PBS. The infected cells were then treated with increasing concentrations of KalsomeTM10, Ambisome and amphotericin B for 1 h after which the cells were washed with PBS and then incubated in fresh medium for 72 h post infection. The coverslips were then washed twice with PBS and air dried. After methanol fixation the coverslips were stained with giemsa and the infected cells were counted on a light microscope using oil emersion lenses and the images were recorded and processed using Adobe Photoshop 5.5 software.

Detection of DNA fragmentation in intracellular amastigotes

DNA fragmentation in intracellular amastigotes was analysed by fluorescence microscopy using the In Situ Apotosis Detection Kit (Trevizen, USA). Mouse peritoneal macrophages (1 x 10⁶) were plated in 8-chamber slides (SPL, Korea) and incubated at 37°C in 5% CO2 overnight. Next day unattached cells were washed off and then infected with parasites at 1:20 ratio. After 3 h of incubation the unbound parasites were removed by washing thrice with PBS. The infected cells were then incubated for further 24 h post infection and then treated with KalsomeTM10, Ambisome and amphotericin B for 1h after which the cells were washed with PBS and then incubated in fresh medium for 72 h post infection. The coverslips were then washed twice with PBS and fixed with 3.7% formaldehyde. The slides were then stained using the In Situ Apoptosis Detection Kit and examined using fluorescence microscopy.

Statistical calculations

All data comparisons were tested for significance using two tailed Student’s t-test using GraphPad software; P values <0.05 were considered significant.

Size and size distribution of KalsomeTM10 and Ambisome

Size distribution of liposomal particles is critical to their function [15], therefore, to assess the mean size and size distribution of KalsomeTM10 and Ambisome, NTA measurement with NanoSight LM20 [16] was performed. We found that the mean hydrodynamic diameter of KalsomeTM10 and Ambisome was 119.5± 14.85 nm and 128.0± 1.414, respectively. Moreover, SD of KalsomeTM10 and Ambisome was found to be 56.50± 14.85 and 53.00± 8.485, respectively, indicating that both these liposomal formulations have similar size distribution pattern in addition to their mean size in the range of nm. Numbers representing average values ± standard deviation (n = 2 measurements) (S1 Fig).

KalsomeTM10 induces killing of L. donovani promastigotes but not macrophages in a time- and concentration-dependent manner

We then investigated the time and dose dependent effect of KalsomeTM10 on promastigotes and macrophages. Treatment of promastigotes for a particular time point with increasing doses of KalsomeTM10 ranging from 0.5–10.0 μg/ml showed a gradual reduction in the cell viability (Fig 1a). Moreover, when the incubation time was increased from 2 to 8 h, drug dose needed for effective killing of the parasite also decreased (Fig 1b). It was found that more than 50% of cells lost viability when treated with 7.5 μg/ml of drug at 2h, which was comparable to those treated with 5.0μg/ml and 2.5 μg/ml of the drug at 4h and 6h, respectively. This suggests that KalsomeTM10 treatment exhibits a time- and concentration- dependent killing of L. donovani promastigotes. To assess the effect of KalsomeTM10 on mammalian cells, we treated mouse peritoneal macrophages with increasing doses of the drug for 48h and 72h, and measured cell viability. Interestingly, we found no significant killing of macrophages at any of the drug doses used (Fig 1c). Taken together, these results suggest that KalsomeTM10 is efficient in killing L. donovani promastigotes with no toxic effect on mammalian cells.

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Fig 1. Effect of KalsomeTM10 on the viability of L. donovani promastigotes and macrophages.

(a) Promastigotes were treated with increasing concentrations of KalsomeTM10 at different time points (2, 4, 6 and 8 h) and the percentage cell viability was estimated. (b) The ED₅₀ and ED₉₀ were determined for each time point. (c) Percent cell viability of murine peritoneal macrophages treated with increasing drug concentrations for 24h and 48h was determined. Results are presented as means±SD; n = 3.

https://doi.org/10.1371/journal.pone.0171306.g001

Leishmanicidal effect of KalsomeTM10 in infected macrophages is dose dependent

Macrophages are the primary host cells, in which Leishmania parasites reside and multiply in the form of amastigotes [17]. Therefore, to assess the efficacy of KalsomeTM10, we treated L. donovani infected macrophages with increasing doses of the drug and determined the infectivity index (no. of amastigotes/100 macrophages) after 72 h of incubation. We found that KalsomeTM10 exhibits a significant killing of intracellular parasites as revealed by decreasing number of amastigotes in infected macrophages, which was totally cleared of at the highest dose used (Fig 2a). The same was represented as Giemsa stained macrophages where progressive reduction in the no. of intracellular parasites proportionate to the increasing dose of KalsomeTM10 treatment was observed (Fig 2b). The phase contrast micrographs of infected macrophages treated with higher concentrations of KalsomeTM10 has also been shown depicting no much difference in the morphology of the infected macrophages compared to the drug treated infected macrophages (S4 Fig) These results suggest that KalsomeTM10 has potent anti-leishmanial activity against intracellular infection model of VL.

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Fig 2. In-vitro leishmanicidal effect of KalsomeTM10 on peritoneal macrophages infected with promastigotes of L. donovani.

Macrophages, infected with promastigotes, were treated with different concentrations of KalsomeTM10 for 72 h post infection. The infected macrophages on coverslips were geimsa stained and counted on a light microscope (a). Number of amastigotes per 100 macrophages was represented as bar graph. Results are presented as means + SD; n = 3. Infected macrophages untreated (b i) and treated with 10 ng/ml (b ii), 50 ng/ml (b iii), 100 ng/ml (b iv), 500 ng/ml (b v) and 1000 ng/ml (b vi) of KalsomeTM10, were giemsa stained and imaged on a light microscope. Pictorial representation of three independent experiments is depicted.

https://doi.org/10.1371/journal.pone.0171306.g002

KalsomeTM10 induces efficient killing of L. donovani promastigotes and intracellular amastigotes compared to Ambisome and amphotericin B

We then compared the in vitro activity of KalsomeTM10 with Ambisome and amphotericin B, taken as control drugs, against promastigotes and intracellular amastigotes in peritoneal macrophages. Promastigotes and intracellular amastigotes were treated with different doses of these drugs for 4h and 72 h respectively, and ED₅₀ and ED₉₀ was determined. KalsomeTM10 showed 50 percent (4.73±0.1282 μg/ml, 0.364±0.0140) and 90 percent (7.87±0.2310 μg/ml, 0.6670±0.043) killing of promastigotes and intracellular amastigotes respectively, at a dose of almost half than that required by Ambisome with ED₅₀ (7.548±0.1189, 0.862±0.0225) and ED₉₀ (13.59±0.2140, 1.552±0.0404), respectively. Although amphotericin B showed 50 percent (0.257±0.0142 μg/ml) and 90 percent (0.437±0.0254) killing of promastigote forms at very low dose, it requires higher concentrations, ED₅₀ (2.00±0.0230) and ED₉₀ (3.609±0.024), respectively, to exhibit intracellular killing of the parasites compared to KalsomeTM10 with ED₅₀ (0.364±0.0140) and ED₉₀ (0.6670±0.043), respectively. Moreover, amphotericin B exhibits toxic effect towards macrophages with increasing doses compared to KalsomeTM10 and Ambisome (Table 1).

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Table 1. KalsomeTM10, Ambisome and amphotericin B differentially affect the viability of L. donovani parasites and the mammalian macrophages.

https://doi.org/10.1371/journal.pone.0171306.t001

Determination of cellular morphology in L. donovani promastigotes

We next investigated the mechanism of cell death in KalsomeTM10 treated promastigotes. Morphological changes are one of the key hallmarks that distinguishes apoptotic and necrotic mode of cell death [18]. So, we initially studied the morphological changes in promastigotes treated with different concentrations of KalsomeTM10 at 4 h. Phase contrast micrographs depicted a significant reduction in size and shape of promastigotes along with substantial loss of flagella, at 1.0 μg/ml of drug treatment compared to the untreated controls (Fig 3a and 3b). Promastigotes treated with 2.5μg/ml and 5.0 μg/ml of the drug, displayed complete rounding in shape along with cytoplasmic condensation and shrinkage (Fig 3c and 3d). In addition, SEM analysis also showed a decrease in cell size, loss of flagella, acquisition of ovoid and irregular shape and severe distortion in cell membrane in KalsomeTM10 treated parasites compared to the untreated control, which possessed elongated morphology with anterior flagella and smooth membrane (Fig 4). These observations suggest that KalsomeTM10 induces significant apoptotic-like morphological changes in promastigotes of L. donovani.

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Fig 3. Cellular morphology of KalsomeTM10 treated L. donovani promastigotes.

Phase contrast micrographs of promastigotes after exposure to KalsomeTM10 for 4 h. Untreated control (a), promastigotes exposed to 1.0 μg/ml (b), 2.5 μg/ml (c) and 5.0 μg/ml (d). Images are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0171306.g003

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Fig 4. Scanning electron microscopy of promastigotes treated with KalsomeTM10.

Promastigotes were either left untreated (a) or treated with 5.0 μg/ml of Kalsome TM10 (b) for 2 h and then analysed for surface topology. SEM micrographs show rounded and distorted shape in KalsomeTM10 treated promastigotes compared to controls. Scale bars: 5 μM. Images are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0171306.g004

KalsomeTM10 induces externalisation of membrane-associated phosphatidylserine (PS) in promastigotes of L. donovani

In the early phase of cellular apoptosis plasma membrane loses asymmetry. This leads to the translocation of phosphatidylserine (PS) from inner to the outer leaflet [19]. Annexin V is a protein which binds to PS. Conversely, PI is a non-permeable dye which selectively enters necrotic cells where it binds to nucleic acids [20]. Therefore, Annexin V-FITC and PI were simultaneously used to assess whether KalsomeTM10 exhibits apoptotic or necrotic mode of cell death in L. donovani promstigotes. For this, flow cytometry was employed which can distinguish cells undergoing early apoptosis [annexin V (+), PI (-)], late apoptosis [annexin V (+), PI (+)], necrosis [annexin V (-), PI (+)] and those that still remained alive [annexin V(-), PI(-)]. Treatment of promastigotes with KalsomeTM10 at 1 h showed a dose-dependent increase in the percentage of early apoptotic cells [annexin V (+), PI (-)] from 20.0% (2.5 μg/ml) to 29.7% (5.0 μg/ml) compared to the untreated control. However, at 2 h treatment, although early apoptotic cells decreased from 28.7% (2.5 μg/ml) to 20.0% (5.0 μg/ml), a simultaneous increase in the percentage of late apoptotic cells [annexin V (+), PI (+)] from 37.8 (2.5 μg/ml) to 45.5% (5.0 μg/ml) were observed, which indicated that with increase in drug exposure time, cells undergo into the late phase of apoptosis. Interestingly, no significant necrotic cells [annexin V (-), PI (+)] were observed (Fig 5). As a control, when promastigotes were treated with Ambisome (7.5 μg/ml) and amphotericin B (0.25 μg/ml) at 1h it showed 8.7% and 28.3% of early apoptotic cells [annexin V (+), PI (-)], respectively compared to the untreated control (S2 Fig). These results strongly indicate that KalsomeTM10 exhibits an apoptotic-like mode of cell death by inducing externalisation of membrane PS in promastigotes of L. donovani.

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Fig 5. PS externalization in KalsomeTM10 treated promastigotes.

Promastigotes, untreated (a and b) and treated with 2.5 μg/ml (c) and 5.0 μg/ml (d) of KalsomeTM10 for 1 h, and treated with 2.5 μg/ml (e) and 5.0 μg/ml (f) of KalsomeTM10 for 2 h, were co stained with annexin V-FITC and PI, and analysed by flow cytometry. The dot plots are representative of one of the three independent experiments. (g) Bar graphs representing mean % PS⁺/PI⁻ cells.**P<0.001.

https://doi.org/10.1371/journal.pone.0171306.g005

KalsomeTM10 induces mitochondrial dysfunction by depolarizing mitochondrial membrane potential (Δѱ_m) and disrupting mitochondrial integrity with a major impact on depletion of cellular ATP pool in Leishmania parasites

Altered mitochondrial function during apoptosis is linked to a change in the mitochondrial membrane potential [21]. JC-1, a cationic, lipophilic and Δѱ_m sensitive dye, is used to measure such change. In intact mitochondria, JC1, in the form of molecular aggregates, emits red fluorescence, but with depolarisation of mitochondrial membrane potential, it comes out in the cytoplasm, in monomeric form, and emits green fluorescence. This results in the decrease in red/green fluorescence intensity ratio [22]. Thus, to verify the effect of KalsomeTM10 on mitochondrial dysfunction, we monitored mitochondrial membrane potential through flow cytometry. We found that KalsomeTM10 exhibits a dose-dependent decrease in the red/green fluorescence intensity ratio compared to the untreated control at 1h of treatment. This was evident from the percentage of cells in the red region and the green region (36.7% and 59.8% for untreated control, 7.7% and 80.7% when treated with 2.5μg/ml KalsomeTM10 and 3.0% and 87.3% when treated with 5.0 μg/ml of KalsomeTM10, respectively) (Fig 6ai–6aiii). In order to further confirm the involvement of mitochondrial apoptotic pathway in KalsomeTM10 treated promastigotes, we assessed the physical integrity of mitochondria using MitoTracker Deep Red (a stain which enters only actively respiring mitochondria in live cells). Flow cytometry analyses showed that the percentage of Mitotracker positive cells decreased in a time-dependent manner, when promastigotes were treated with 2.5μg/ml of KalsomeTM10 (Fig 6bi–6biii). This could be observed in the form of Mean fluorescence intensity (MFI), whose value fell drastically in the treated cells compared to the untreated control (Fig 6biv). Based on these results, we sought to determine, whether KalsomeTM10 affects the intracellular pool of ATP, as mitochondria are the major contributors to ATP generation, and any disruption in the mitochondrial structure and function would directly affect the intracellular ATP levels [23]. To address this, we treated promastigotes with different doses of KalsomeTM10 at 4 h and measured ATP content using a bioluminiscence kit (Invitrogen). Our results showed that KalsomeTM10 induces a dose-dependent depletion in the total intracellular ATP content, which was to the extent of 27%, 60%, 72% and 80% at 1, 2.5, 5.0 and 7.5 μg/ml of KalsomeTM10 treatment respectively, compared to the untreated control (Fig 6c). Collectively, these results suggest that KalsomeTM10 causes a major mitochondrial dysfunction by inducing depolarisation of mitochondrial membrane potential, loss of mitochondrial integrity and depletion of cellular ATP pool in promastigotes of L. donovani.

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Fig 6. Effect of KalsomeTM10 on mitochondrial membrane potential (Δѱ_m), mitochondrial integrity and intracellular ATP levels.

Promastigotes, untreated (a i) and treated with 2.5 μg/ml (a ii) and 5.0 μg/ml (a iii) of KalsomeTM10 for 1 h, were stained with potentiometric probe JC-1, and analysed by flow cytometry. The dot plots are representative of three independent experiments. Bar graphs representing mean % green flourescent cells (a iv). Promastigotes untreated (b i) and treated with 2.5 μg/ml of KalsomeTM10 for 1 h (b ii) and 2 h (b iii), were stained with mitotraker, and analysed by flow cytometry. Histograms are representative of three independent experiments with fold change depicted as MFI (b iv). Promastigotes were incubated with KalsomeTM10 for 4 h. Cellular ATP concentration was then measured by bioluminescence assay. ATP concentration was expressed as nM (c). Results are presented as means + SD; n = 3. *P<0.05, **P<0.001, ***P<0.0001.

https://doi.org/10.1371/journal.pone.0171306.g006

KalsomeTM10 leads to the generation of intracellular reactive oxygen species (ROS) and lipid peroxidation products in promastigotes of L. donovani

Oxidoredox homeostasis is essential for cellular survival. During apoptosis altered mitochondrial function results in overproduction of ROS that leads to an oxidative stress and cellular damage [24]. To investigate whether KalsomeTM10 induces ROS generation in leishmanial cells, we measured cellular H₂O₂ using a bioluminescence kit (promega). We found that KalsomeTM10 facilitates a dose- (2.5 and 5.0 μg/ml) and time- (0.5 to 2 h) dependent increase in H₂O₂ production which however, diminished progressively, when the treatment was carried out in presence of increasing concentrations of the ROS scavenger N-acetyl-L-cysteine (NAC) (1mM and 10 mM) (Fig 7ai and 7aii). We also studied the effect of KalsomeTM10 on mitochondrial superoxide (O₂⁻) production through flow cytometry. Again, our results showed a dose- and time-dependent increase in mitochondria O₂⁻ production in leishmanial cells with no such production in untreated control (Fig 7b and 7c). With these results, we sought to verify, whether KalsomeTM10 treatment has any effect on the cellular pool of glutathione (GSH), since redox status in cells is under the tight control of GSH, a potent antioxidant molecule [25]. We measured GSH content in KalsomeTM10 (2.5 μg/ml) treated promastigotes using a flourometric assay kit (Biovision) and found that there was a marked depletion in GSH pool in drug treated parasites compared to the untreated controls (Fig 7d).

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Fig 7. ROS and mitochondrial super-oxide production, and depletion of GSH pool in KalsomeTM10 treated promastigotes.

Promastigotes incubated at different time points with KalsomeTM10 at 2.5 μg/ml (a i) and 5.0 μg/ml (a ii), with or without NAC (1 mM and 10 mM). Cellular ROS was measured by bioluminescence assay. Results are presented as means + SD; n = 3. Promastigotes, untreated (b i and ii) and treated with 2.5 μg/ml of KalsomeTM10 for 30 min (b iii) and 1 h (b iv), and treated with 5.0 μg/ml of KalsomeTM10 for 30 min (b v) and 1 h (b vi), were stained with mitosox, and analysed by flow cytometry. The histograms are representative of three independent experiments. (c) Bar graphs representing mean % superoxide⁺ cells. (d) Promastigotes treated with 2.5 μg/ml of KalsomeTM10 at different time points and GSH concentration determined flourometrically. Results are presented as means + SD; n = 3. *P<0.01, **P<0.001.

https://doi.org/10.1371/journal.pone.0171306.g007

Lipid peroxidation, a process in which oxidants such as free radicals target polyunsaturated fatty acids resulting in lipid peroxyl radicals and hydroperoxides generation, is detrimental to cells leading to their death either through apoptotic or necrotic mode [26]. Thus, to assess whether KalsomeTM10 induces generation of lipid peroxidation products, we treated promastigotes at 2 h with varying doses of the drug and measured lipid peroxidation products flourometrically. Our results showed that KalsomeTM10 induces a dose-dependent increase in these products (Fig 8a). To further confirm that KalsomeTM10 does so by generating ROS, we treated promastigotes with KalsomeTM10 in the presence of NAC. The results showed diminished production of lipid peroxidation products in the presence of NAC, indicating that KalsomeTM10 exhibits potent ROS generation in promastigotes that leads to the production of these products (Fig 8b). Altogether, these observations suggest that KalsomeTM10 mediated mitochondrial dysfunction results in over production of free radicals with concomitant depletion of intracellular GHS content and increased generation of lipid peroxidation products, which triggers apoptotic-like mode of cell death in promastigotes.

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Fig 8. Generation of lipid peroxidation products on treatment with KalsomeTM10.

Promastigotes were treated with different concentrations of kalsomeTM10 for 2 h in the absence (a) and presence of NAC (b), and lipid peroxidation products were determined flourometrically. Results are presented as means + SD; n = 3. P<0.05.

https://doi.org/10.1371/journal.pone.0171306.g008

Induction of in vivo caspase-like activity in L. donovani by KalsomeTM10

Caspases are one of the key molecules which orchestrate the process of apoptosis [27]. Thus to determine, if caspases really play any role in KalsomeTM10 mediated cell death in promastigotes, we used Apostat (a cell permeable caspase specific substrate with flourescent tag; from R&D), which detects intracellular active caspase. Flow cytometric analyses showed that KalsomeTM10 treatment induces a dose- and time- dependent increase in caspase positive cells, which was significantly reduced by the pan caspase inhibitor, z-VAD-fmk, suggesting that KalsomeTM10 activates caspase mediated apoptotic machinery in promastigotes of L. donovani (Fig 9a). To further confirm the involvement of caspases, we performed cell viability assay in the presence and absence of z-VAD-fmk. Our results showed that z-VAD-fmk significantly reduced cell death in promastigotes at 1h treatment of KalsomeTM10 (2.5 μg/ml) (Fig 9b), which also reflected in the decrease in the percentage of annexin-v positive cells (Fig 9c). Treatment of promastigotes with Ambisome (7.5 μg/ml) and amphotericin B (0.25 μg/ml) at 1h also showed induction of caspase positive cells (S3 Fig). These results suggest that KalsomeTM10 mediated cell death in promstigotes of L. donovani is caspase dependent.

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Fig 9. Evaluation of caspase activity in KalsomeTM10 treated L. donovani promastigotes.

Promastigotes were either left untreated (a i and ii) or treated with 2.5 μg/ml (a iii) and 5.0 μg/ml (a iv) of kalsomeTM10 for 30 min and treated for 1h in absence or presence of z-VAD-fmk with 2.5 μg/ml (a v and vi) and 5.0 μg/ml (a vii and viii) of kalsomeTM10, respectively. Caspase positive cells were analysed through flow cytometry and plotted as histograms which are representative of three independent experiments. (a ix) Mean caspase positive cells represented as bar graphs. (b) Percent cell viability was determined in promastigotes treated with different concentrations of KalsomeTM10 for 2 h in the absence and presence of z-VAD-fmk. Results are presented as means + SD; n = 3. Promastigotes, either untreated (c i) or treated with 2.5 μg/ml kalsomeTM10 for 1 h in the absence (c ii) and presence of z-VAD-fmk (c iii), were co stained with annexin V-FITC and PI, and analysed by flow cytometry. The dot plots are representative of three independent experiments. (c iv) Mean PS⁺/PI⁻ cells represented as bar graphs. *P<0.01.

https://doi.org/10.1371/journal.pone.0171306.g009

Determination of apoptotic-like cell death by oligonucleosomal DNA fragmentation in L. donovani promastigotes

The cleavage pattern of genomic DNA characterized by fragmentation into nucleosomal units is a distinctive feature of apoptotic cell death [28]. To investigate whether KalsomeTM10 exhibits DNA fragmentation in L. donovani promastigotes, we performed TUNEL staining that detects fragmented DNAs by labelling their free ends with dUTP. We found that KalsomeTM10 significantly increased TUNEL positive cells in a dose-dependent manner compared to the untreated control cells (Fig 10a). To further substantiate this observation, we also performed DNA laddering assay, by employing agarose gel electrophoresis. The results showed that there was marked fragmentation in genomic DNA into oligonucleosome-sized fragments (in multiples of 200 bp) when promastigotes were treated with 1.0 μg/ml (lane 3), 2.5 μg/ml (lane 4), and 5.0 μg/ml (lane 5) of KalsomeTM10. In contrast, untreated cells (lane 2) did not show any DNA fragmentation (Fig 10b). Moreover, it is evident from Fig 10b that DNA laddering initiated at the lowest drug dose and with further increase in the dose, there was a marked decrease in the size of the intact DNA with a corresponding increase in the DNA laddering when compared to the untreated controls. Overall, these results suggest that KalsomeTM10 causes genomic DNA fragmentation, which might contribute to the induction of apoptotic-like cell death in L. donovani promastigotes.

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Fig 10. Fragmentation of genomic DNA in KalsomeTM10 treated promastigotes analysed through TUNEL assay and DNA laddering assay.

Promastigotes were either left untreated (a i) or treated with 1.0 μg/ml (a ii) 2.5 μg/ml (a iii) and 5.0 μg/ml (a iv) of KalsomeTM10 for 12 h, and then TUNEL assay was performed. The fluorescence was analysed by flow cytometry and presented as histograms which are representative of two independent experiments. (b) Mean TUNEL+ cells represented as bar graphs. (c) Promastigotes treated with different concentrations of KalsomeTM10 for 14 h. Apoptotic DNA was resolved on a 1.5% agarose gel. Representative images of three independent experiments. *P<0.01, **P<0.001.

https://doi.org/10.1371/journal.pone.0171306.g010

KalsomeTM10 induces cell cycle arrest in L. donovani promastigotes

Degradation of genomic DNA has profound effects on the replication of cells [29]. To verify whether KalsomeTM10 affects promastigote’s replication, we treated parasites with different doses of the drug at varying time points, and analysed cell cycle progression through flow cytometry. The results indicated that KalsomeTM10 induces a dose- and time-dependent arrest of leishmanial cells, since the percentage of cells in the sub-G0 phase of the cell cycle increased when compared to the untreated control (Fig 11a and 11b).

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Fig 11. Analysis of cell cycle arrest in L. donovani promastigotes treated with KalsomeTM10.

Promastigotes, either left untreated (a i) or treated with 2.5 μg/ml (a ii) and 5.0 μg/ml (a iii) of kalsomeTM10 for 2 h and untreated (b i) or treated with 2.5 μg/ml (b ii) and 5.0 μg/ml (b iii) of KalsomeTM10 for 4 h, were analysed for cell cycle through flow cytometry and plotted as histograms which are representative of three independent experiments. (c) Mean % Sub G0 cells represented as bar graphs.**P<0.001, ***P<0.0001.

https://doi.org/10.1371/journal.pone.0171306.g011

KalsomeTM10 induces apoptotic DNA fragmentation in intracellular amastigotes of L. donovani

To determine the mechanism of killing of intracellular amastigotes, peritoneal macrophages were infected with stationay phase promastigotes of L. donovani. After 24h of incubation, infected macrophages were treated with KalsomeTM10 and control drugs Ambisome and amphotericin B for 48h. TUNEL assay show that compared to untreated infected macrophages (Fig 12a) KalsomeTM10 treatment induces DNA degradation into apoptotic fragments as revealed by prominent green signal in the amastigotes without affecting macrophage DNA (Fig 12b). Ambisome (Fig 12c) and amphotericin B (Fig 12d) also induced DNA fragmentation but with low efficacy compared to KalsomeTM10 as revealed by the moderate green signal intensity.

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Fig 12. Analysis of genomic DNA fragmentation in KalsomeTM10, Ambisome and amphotericin B treated intracellular amastigotes analysed through TUNEL assay.

L. donovani infected murine peritoneal macrophages were either left untreated (a) or treated with KalsomeTM10 (b) at 0.5 μg/ml, Ambisome (c) at 1.0 μg/ml and amphotericin B (d) at 2.0 μg/ml. After 48 h, infected macrophages were stained using TUNEL and analysed by fluorescence microscopy. Total nuclei are visualized in red (PI) and TUNEL-positive nuclei are stained in green (FITC). The images are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0171306.g012

KalsomeTM10 mediated killing of intracellular amastigotes is ROS and nitric oxide dependent

ROS and nitric oxide are potent anti-leishmanial molecules [30,31]. Therefore, to verify if kalsomeTM10 induces generation of these molecules as a probable mechanism of killing intracellular amastigotes, peritoneal macrophages were infected with promastigotes of L. donovani. After 24 h of incubation, infected macrophages were treated with KalsomeTM10 in absence and presence of the ROS scavangers (NAC, PEG-Cat and PEG-SOD) and nitric oxide synthesis inhibitor (L-NMMA) for 48 h and the infectivity index (no. of amastigotes/100 macrophages) was determined. We found that KalsomeTM10 mediated killing of intracellular parasites was significantly inhibited in the presence of the above mentioned ROS scavengers and nitric oxide synthesis inhibitor revealing that KalsomeTM10 employs these antimicrobial molecules to kill the parasites (Fig 13).

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Fig 13. Role of ROS and nitric oxide generation on in-vitro leishmanicidal effect of KalsomeTM10 on intracelluar amsastigotes.

Macrophages, infected with promastigotes, for 24 h were pretreated with ROS scavangers like NAC (10 mM), PEG-Cat (500 units) and PEG-SOD (500 units) and nitric oxide inhibitor L-NMMA (2 mM) for 30 mins and then treated with KalsomeTM10 (0.5 μg/ml) for 1h. After washing, incubation for 48 h post drug treatment agan in the prescence of these scavangers and inhibitor was carried out and then the infected macrophages were geimsa stained and counted on a light microscope to determine infectivity index (Number of amastigotes per 100 macrophages) that was represented as bar graph. Results are presented as means + SD; n = 2.***P<0.0001.

https://doi.org/10.1371/journal.pone.0171306.g013

Endocytosis play important role in KalsomeTM10 mediated but not amphotericin B mediated killing of intracellular amastigotes

Endocytosis of liposomal formulations to cells is crucial to their function [32,33]. Therefore, to evaluate if endocytosis is necessary for kalsomeTM10 and amphotericin B mediated anti-leishmanial effects, peritoneal macrophages were infected with promastigotes of L. donovani. After 24 h of incubation, infected macrophages were pretreated with cytochalasin D for 1h and then treated with KalsomeTM10 and amphotericin B for 1h. After washing the cells, further incubation for 48 h was carried out and the infectivity index (no. of amastigotes/100 macrophages) was determined. We found that KalsomeTM10 mediated killing of intracellular parasites was significantly inhibited in the presence of the cytochalasin D but no such inhibition was observed in amphotericn B treated macrophages, indicating that endocytosis has crucial role in KalsomeTM10 mediated anti-leishmanial effect (Fig 14a).

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Fig 14. Effect of cytochalasin D and MβCD on in-vitro leishmanicidal effect of KalsomeTM10 and amphotericin B on intracelluar amsastigotes.

Macrophages, infected with promastigotes, for 24 h were pretreated with cytochalasin D (25 mM) (a) and MβCD (20 mM) (b) for 30 mins and then treated with KalsomeTM10 (0.5 μg/ml) and amphotericin B (3 μg/ml) for 1h in cytochalasin D treted macrophage and for 30 mins in MβCD treated macrophages. After washing, incubation for 48 h post drug treatment in the prescence of these scavangers and inhibitor was carried out and then the infected macrophages were geimsa stained and counted on a light microscope to determine infectivity index (Number of amastigotes per 100 macrophages) that was represented as bar graph. Results are presented as means + SD; n = 2. ***P<0.0001.

https://doi.org/10.1371/journal.pone.0171306.g014

Cholesterol depletion on host macrophage diminished KalsomeTM10 and amphotericin B mediated killing of intracellular amastigotes

Amphotericin B binds to membrane cholesterol [32], therefore, to determine if cholesterol depletion of macrophage affects the leishmanicidal function of kalsomeTM10 and amphotericin B, peritoneal macrophages were infected with promastigotes of L. donovani. After 24 h of incubation, infected macrophages were pretreated with MβCD for 40 mins and then treated with KalsomeTM10 and amphotericin B for 1h. After washing the cells, further incubation for 48 h was carried out and the infectivity index (no. of amastigotes/100 macrophages) was determined. We found that not only amphotericn B but also KalsomeTM10 mediated killing of intracellular parasites was significantly inhibited in cholesterol depleted macrophages, indicating that membrane cholesterol plays crucial role in KalsomeTM10 and amphotericn B mediated anti-leishmanial effect (Fig 14b).