# In Vitro Antiparasitic and Apoptotic Effects of Antimony Sulfide Nanoparticles on Leishmania infantum

Authors: Saied Soflaei; Abdolhossein Dalimi; Fatemeh Ghaffarifar; Mojtaba Shakibaie; Ahmad Reza Shahverdi; Mohsen Shafiepour
Journal: Journal of Parasitology Research
 (2012)

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## Abstract

Visceral leishmaniasis is one of the most important sever diseases in tropical and subtropical countries. In the present study the effects of  antimony sulfide nanoparticles onLeishmania infantum in vitro were evaluated. Antimony sulfide NPs (Sb2S5) were synthesized by  biological method from Serratia marcescens bacteria. Then the cytotoxicity effects of different concentrations (5, 10, 25, 50, and 100 μg/mL) of this nanoparticle were assessed on promastigote and amastigote stages of L. infantum. MTT method was used for verification results of promastigote assay. Finally, the percentages of apoptotic,  necrotic, and viable cells were determined by flow cytometry. The results indicated the positive effectiveness of antimony sulfide NPs on proliferation of promastigote  form. The IC50 (50% inhibitory concentration) of antimony sulfide NPs on promastigotes was calculated 50 μg/mL. The cytotoxicity effect was dose-dependent means by increasing the concentration of antimony sulfide NPs, the cytotoxicity curve was raised and the viability curve of the parasite dropped simultaneously. Moreover, the IC50 of antimony sulfide NPs on amastigote stage was calculated 25 μg/mL. On the other hand, however, antimony  sulfide NPs have a low cytotoxicity effect on uninfected macrophages but it can  induce apoptosis in promastigote stage at 3 of 4 concentrations.


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## Body

## 1. Introduction

Leishmaniasis is considered as one of the most important tropical diseases with worldwide distribution [1]. The  disease  is  reported  in  88  countries  around  the  world, and  its  prevalence  is  estimated  to  be  approximately  12  million  annually  and  about  350  million  people  are  at  the  risk  of  the  disease [1, 2]. About 90% of cases of cutaneous leishmaniasis are found in Brazil, Afghanistan, Iran, Peru, Saudi Arabia, and Syria, and about 90% cases of visceral leishmaniasis are reported in Bangladesh, Brazil, Nepal, India, and Sudan [3, 4]. Visceral  leishmaniasis  (Kala-azar)  is  characterized  by  the  presence  of  fever, splenomegaly, hepatomegaly, swollen  lymph  nodes,  and  weight  loss  that  depends  on  the  pathogenicity  of  Leishmania  species  and  the  host  immune  response against  parasite [5, 6].  About  90%  of  the cases of  this  disease may  lead  to  death  if  it  is left without any treatment. Leishmaniasis coinfection  with  HIV  and  other  immunosuppression  is  becoming  another  serious  problem, therefore  the  treatment  methods  mostly  focus  on  induction  of  immune  responses  [5]. Pentavalent antimonials are a group of compounds used for the treatment of leishmaniasis. The compounds currently available for clinical use are sodium stibogluconate (Pentostam) and meglumine antimonate (Glucantime). In  systemic  therapy  of  leishmaniasis these  drugs  are  used  alone  or  in  combination  with  other compounds [7–15]. The current  drugs  is not so much suitable due to resistance reported, high  toxicity,  various  side  effects and so forth.  So,  new  therapeutic antileishmanial strategies  are  urgently  required  [5, 16].Nanomedicine is the medical application of nanotechnology. Nanomedical approaches to drug delivery center on developing nanoscale particles [17]. Up to now various nanoparticle compounds have been introduced against leishmaniasis [18–27]. In  the  present study  we  evaluated the effects  of  antimony  sulfide  nanoparticles  on  Leishmania infantum in vitro.


## 2. Materials and Methods

### 2.1. Drug Preparation

Antimony  sulfide  nanoparticle was synthesized  by  intracellular  biological  methods  from  nonpigmentedby using of the Serratia marcescens bacterial isolate from the Caspian Sea in northern of Iran  with  the  size  less  than  35 nm  according  to  Bahrami et al. [28] in  the Department  of  Pharmaceutical  Biotechnology  and  Pharmaceutical  Sciences  Research  Centre,  Faculty  of  Pharmacy, Tehran  University  of  medical  sciences.


### 2.2. Parasites

Leishmania  infantum  MON-1  (MHOM/TN/80/IPT1)  was  provided  from  Pasteur  Institute  of  Iran.  Promastigotes  were  cultured  in  RPMI1640  medium  supplemented  with  10%  fetal  calf  serum  (FCS)  and  antibiotics  (100 IU/mL of penicillin and 100 μg/mL  of  streptomycin). The culture was maintained in 24°C for promastigote proliferation. The parasites were transferred weekly from previous culture into new medium.


### 2.3. Drug Assessment

The  interaction  of  antimony  sulfide  NPs  directly  and  promastigotes  was  studied. After  proliferation  of the parasites, 100μL of promastigotes  (2 × 106 cell/mL)  was  seeded  in  24-well  plate  containing  100 μL of RPMI1640 medium  and  treated  with  serial  dilutions  of  the  antimony  sulfide NPs (5, 10, 25, 50, and 100 μg/mL) for 24, 48, and 72 hours. After incubation, the antileishmanial activity  of  antimony  sulfide  NPs  was evaluated by  direct  counting of parasites.  These  data  were  analyzed  by  Graph  pad  Prism  version  5.04  software.


### 2.4. MTT Test

Briefly, 100μL  of  promastigotes  (2 × 106 cells/mL)  was  cultured  separately  in  96-well  microplates  containing  100 μL  of  RPMI1640  medium  supplemented  with  20%  FCS.  These  cultures  were  repeated  at  least  three  times  in  triplicate  wells. 200 μl of promastigotes  were  cultured  as  control  group. 200 μL/well  PBS  was  added  around  well  of  plates  to  prevent  the  evaporation  of  well  contents. The  cells  were  incubated  in  presence  seven  dilutions  of  antimony  sulfide  NPs  at  24±1°C  for  72  hours  and  then  20 μL  of  MTT  solution  was  added  into  each  of  wells.  Plates  were  incubated  again  at  24°C  for  4  hours  and  then  centrifuged  at  1000 g for 10  minutes.  Supernatant was aspirated gently and discarded.  100 μL  DMSO  was  added  to  each  of  the wells  and  finally  the  absorbance  of  these  plates  was  measured  by  the  ELISA  reader  system  in  at  540 nm.


### 2.5. Macrophage Cytotoxicity Measurement

Inbred  male  BALB/c  mice  were  prepared  from  Razi  Institute  of  Iran. The effect of antimony sulfide  NPs  on  macrophages of infected and uninfected mice was evaluated. In this regard, 7 mL of RPMI medium (sigma) was injected into peritoneumand macrophages were collected.  Then the number of live macrophages was counted.  100μL  of  macrophages  with  100 μL  RPMI1640  medium  were  seeded  in  exposure  to  seven  dilutions  of  antimony  sulfide  NPs.  These  cultures  were  maintained  at  37°C  in  the  presence  of  5%  CO2  for  24,  48, and  72  hours.  The experiment was terminated by direct counting. Cytotoxic  effect  of  antimony  sulfide  NPs  on  macrophage was evaluated and  compared  with  control  cultures.


### 2.6. Intracellular  Amastigote  Assay

Peritoneal  cavity  macrophages  of BALB/c  were  seeded  in  24-well  plates  and  incubated  at 37°C with  5% CO2 for 24 hours for differentiation. The cells were infected with promastigotes of stationary growth phase at a parasite/macrophage ratio of 10 : 1. Drug susceptibilities of intracellular amastigotes were assessed with the method previously described by Tada et al. [29]. The culture  was  incubated  at  37°C  in  the  presence  of  5%  CO2  for  24  hours  until  promastigotes  were  phagocyte  by  macrophages. After  incubation, each  well  of  the  plates  was  washed  with  1-2 mL  PBS  to remove  the  extracellular  promastigotes. Then infected macrophages  were  separated  from  the  plates  by  cold  method  (10–15  minutes  on  ice  pieces).  Then  5 μL  of  these  cells  were  stained  by  Giemsa  method. The percentage of infected cells and the number of amastigotes in each cell was microscopically assessed.  After that  100 μL  of  these  cells  were  transferred  into  new  plate  and  were  incubated  with  seven  dilutions  of  antimony  sulfide  NPs  at  37°C  with  5% CO2  for  24,  48, and 72  hours.  Finally, the  plates  were  incubated  on  ice  pieces  for  10–15  minutes. The  percentage  of  infection and  IC50 was calculated  through  examination  of  200  macrophages  and  the  number  of  amastigotes  in  every  single  cell. The results  were  expressed  as  the  infection  index, which is reflecting of drug effect in prevention of infection.


### 2.7. Promastigote  Apoptosis Assessment

At  first  2 × 106cells/ml of promastigotes  were  treated  with  various  dilutions  (10,  25,  50, 100 μg/mL)  of  antimony  sulfide  NPs  in  ELISA  plates  and  incubated  at  24°C  for  72  hours.  Test  and  control  wells  were  washed  twice  by  cold  PBS  solution  and  centrifuged  in  1400 rpm  for 10 min. 100 μL  Annexin-V  FITC  solution  and  100 μL  PI  (propidium  iodide)  solution  were  added  and  incubated  for  15  minutes  at  room  temperature.  Subsequently,  cellular  apoptosis  in  our  study  was  detected  by  using  Annexin-V  FLUOS  staining  kit  (Roche,  Germany). The  procedure  was  performed  according  to  manufacturing  protocol  in  the  dark  place  and  was  evaluated FACSCalibur system.  Afterwards the flow cytometry results were then analyzed using CellQuest software.


## 2.1. Drug Preparation

Antimony  sulfide  nanoparticle was synthesized  by  intracellular  biological  methods  from  nonpigmentedby using of the Serratia marcescens bacterial isolate from the Caspian Sea in northern of Iran  with  the  size  less  than  35 nm  according  to  Bahrami et al. [28] in  the Department  of  Pharmaceutical  Biotechnology  and  Pharmaceutical  Sciences  Research  Centre,  Faculty  of  Pharmacy, Tehran  University  of  medical  sciences.


## 2.2. Parasites

Leishmania  infantum  MON-1  (MHOM/TN/80/IPT1)  was  provided  from  Pasteur  Institute  of  Iran.  Promastigotes  were  cultured  in  RPMI1640  medium  supplemented  with  10%  fetal  calf  serum  (FCS)  and  antibiotics  (100 IU/mL of penicillin and 100 μg/mL  of  streptomycin). The culture was maintained in 24°C for promastigote proliferation. The parasites were transferred weekly from previous culture into new medium.


## 2.3. Drug Assessment

The  interaction  of  antimony  sulfide  NPs  directly  and  promastigotes  was  studied. After  proliferation  of the parasites, 100μL of promastigotes  (2 × 106 cell/mL)  was  seeded  in  24-well  plate  containing  100 μL of RPMI1640 medium  and  treated  with  serial  dilutions  of  the  antimony  sulfide NPs (5, 10, 25, 50, and 100 μg/mL) for 24, 48, and 72 hours. After incubation, the antileishmanial activity  of  antimony  sulfide  NPs  was evaluated by  direct  counting of parasites.  These  data  were  analyzed  by  Graph  pad  Prism  version  5.04  software.


## 2.4. MTT Test

Briefly, 100μL  of  promastigotes  (2 × 106 cells/mL)  was  cultured  separately  in  96-well  microplates  containing  100 μL  of  RPMI1640  medium  supplemented  with  20%  FCS.  These  cultures  were  repeated  at  least  three  times  in  triplicate  wells. 200 μl of promastigotes  were  cultured  as  control  group. 200 μL/well  PBS  was  added  around  well  of  plates  to  prevent  the  evaporation  of  well  contents. The  cells  were  incubated  in  presence  seven  dilutions  of  antimony  sulfide  NPs  at  24±1°C  for  72  hours  and  then  20 μL  of  MTT  solution  was  added  into  each  of  wells.  Plates  were  incubated  again  at  24°C  for  4  hours  and  then  centrifuged  at  1000 g for 10  minutes.  Supernatant was aspirated gently and discarded.  100 μL  DMSO  was  added  to  each  of  the wells  and  finally  the  absorbance  of  these  plates  was  measured  by  the  ELISA  reader  system  in  at  540 nm.


## 2.5. Macrophage Cytotoxicity Measurement

Inbred  male  BALB/c  mice  were  prepared  from  Razi  Institute  of  Iran. The effect of antimony sulfide  NPs  on  macrophages of infected and uninfected mice was evaluated. In this regard, 7 mL of RPMI medium (sigma) was injected into peritoneumand macrophages were collected.  Then the number of live macrophages was counted.  100μL  of  macrophages  with  100 μL  RPMI1640  medium  were  seeded  in  exposure  to  seven  dilutions  of  antimony  sulfide  NPs.  These  cultures  were  maintained  at  37°C  in  the  presence  of  5%  CO2  for  24,  48, and  72  hours.  The experiment was terminated by direct counting. Cytotoxic  effect  of  antimony  sulfide  NPs  on  macrophage was evaluated and  compared  with  control  cultures.


## 2.6. Intracellular  Amastigote  Assay

Peritoneal  cavity  macrophages  of BALB/c  were  seeded  in  24-well  plates  and  incubated  at 37°C with  5% CO2 for 24 hours for differentiation. The cells were infected with promastigotes of stationary growth phase at a parasite/macrophage ratio of 10 : 1. Drug susceptibilities of intracellular amastigotes were assessed with the method previously described by Tada et al. [29]. The culture  was  incubated  at  37°C  in  the  presence  of  5%  CO2  for  24  hours  until  promastigotes  were  phagocyte  by  macrophages. After  incubation, each  well  of  the  plates  was  washed  with  1-2 mL  PBS  to remove  the  extracellular  promastigotes. Then infected macrophages  were  separated  from  the  plates  by  cold  method  (10–15  minutes  on  ice  pieces).  Then  5 μL  of  these  cells  were  stained  by  Giemsa  method. The percentage of infected cells and the number of amastigotes in each cell was microscopically assessed.  After that  100 μL  of  these  cells  were  transferred  into  new  plate  and  were  incubated  with  seven  dilutions  of  antimony  sulfide  NPs  at  37°C  with  5% CO2  for  24,  48, and 72  hours.  Finally, the  plates  were  incubated  on  ice  pieces  for  10–15  minutes. The  percentage  of  infection and  IC50 was calculated  through  examination  of  200  macrophages  and  the  number  of  amastigotes  in  every  single  cell. The results  were  expressed  as  the  infection  index, which is reflecting of drug effect in prevention of infection.


## 2.7. Promastigote  Apoptosis Assessment

At  first  2 × 106cells/ml of promastigotes  were  treated  with  various  dilutions  (10,  25,  50, 100 μg/mL)  of  antimony  sulfide  NPs  in  ELISA  plates  and  incubated  at  24°C  for  72  hours.  Test  and  control  wells  were  washed  twice  by  cold  PBS  solution  and  centrifuged  in  1400 rpm  for 10 min. 100 μL  Annexin-V  FITC  solution  and  100 μL  PI  (propidium  iodide)  solution  were  added  and  incubated  for  15  minutes  at  room  temperature.  Subsequently,  cellular  apoptosis  in  our  study  was  detected  by  using  Annexin-V  FLUOS  staining  kit  (Roche,  Germany). The  procedure  was  performed  according  to  manufacturing  protocol  in  the  dark  place  and  was  evaluated FACSCalibur system.  Afterwards the flow cytometry results were then analyzed using CellQuest software.


## 3. Statistical Analysis

The results of test and control groups were analyzed and compared by ANOVA statistical test (P≤0.05) using SPSS version 15 software and Graph pad prism version 5.04.


## 4. Results

The results indicated the positive  effectiveness  of  antimony  sulfide  NPs  on  proliferation  of  promastigote form. The cytotoxic  effect of 5  dilutions  of  antimony  sulfide  NPs  on  promastigotes  was assessed and compared with control group in Figure1. The IC50 (50% inhibitory  concentration)  of  antimony  sulfide  NPs  on  promastigotes  was calculated 50 μg/ml.Figure 1
Viability  of  test  and  control  groups  of promastigote  ofL.infantum in 5  dilutions  of  antimony  sulfide  NPs during  24,  48,  and  72  hours of incubation.MTT  method  was  used  for  verification  results  of  promastigote  assay.  Cytotoxicity  of different concentration of the drug and  viability  of promastigote stage of the parasite are  shown in  Figure2. By increasing the concentration of antimony  sulfide  NPs, the cytotoxicity curve raised and the viability curve of the parasite dropped simultaneously.Figure 2
Percentage of   viability of promastigotes  ofLeishmaniainfantum  and cytotoxicity of  5  dilutions  of  antimony  sulfide  NPs  at  72  hours  by  MTT  method.Cytotoxic  effect  of  5  dilutions  of  antimony  sulfide  NPs  on  uninfected  splenic  macrophages  of  BALB/c  mice  was  compared  with  control  cultures  at  24,  48,  72  hours.  The result as is  shown  in  Figure3, indicated that  antimony  sulfide  NPs  has  low  cytotoxicity  effect  on  uninfected  macrophages.Figure 3
Viability  of  uninfected  mouse  macrophages  in  5  dilutions  of  antimony  sulfide  NPsin  vitro  conditions  during  24,  48,  and  72  hours of incubation.The viability  of  mouse  macrophages  contained  amastigotes  ofLeishmania  infantum  in  5  dilutions  of  antimony  sulfide  NPs  in  vitro  conditions  during  24,  48,  and  72  hours of incubation is shown in Figure 4. Moreover, the IC50 of  antimony  sulfide  NPs  on  amastigote stage  of L. infantum was  calculated  25 μg/mL.Figure 4
Viability  of  mouse  macrophages  contained  amastigotes  ofLeishmania  infantum  in  5  dilutions of  antimony sulfide NPs in  vitro  conditions  during  24,  48,  and  72  hours of incubation.The percentages of apoptotic, necrotic, and viable cells were determined by flow cytometry.  The  basis  of  4  areas are  the  cells  staining  with  Annexin-V  only  as  apoptotic  cells  (lower  right  region),  the  cells  staining  with  PI  as  necrotic  cells  (upper  left  region),  the  cells  staining  with  both  of  Annexin-V  and  PI  as  late  apoptotic  (upper  right  region),  and  those  cells  that  did  not  stain  as  healthy  cells  (lower  left  region). The  result  indicated that  antimony  sulfide  NPs  can  induce  apoptosis  in  promastigote  stage ofLeishmania  at  3  of  4  concentrations (Figure 5).Induction of apoptosis in promastigotes ofLeishmania infantum evaluated by flow cytometry method. (a)  Most  of  promastigotes  were  alive  and  healthy  (control  group), (b) under 10 μg/mL of  antimony sulfide  NPs,  (c) under  25 μg/mL of  antimony  sulfide  NPs,  (d) under 50 μg/mL of  antimony  sulfide  NPs, and (e) under 100 μg/mL of  antimony  sulfide  NPs.
(a)
(b)
(c)
(d)
(e)


## 5. Discussion

Pentavalent  antimonials including  meglumine  antimoniate  (Glucantime)  and  stibogluconate  sodium  (Pentostam)  are  considered  the  drugs  of  choice  for  treatment  of  all  clinical  forms  of  leishmaniasis,  for  40  years [30–36].  Despite  some limitations  attributed  to  its  use  due to resistance reported, high  toxicity,  various  side  effects  and  the  high  cost,  they  still remain  the  most  important  drugs  against  leishmaniasis.  So, the  need  for new  effective  drugs  with  low  toxicity  and  more  effectiveness is critical.Nanotechnology can be a useful tool for synthesize new drugs against infectious diseases. Nanoparticles like emulsomes, liposomes, and nanospheres have been of great importance for drug delivery as drug carriers [37]. Among several nanoparticles implementing for treatment, liposomes are the best for evaluating the efficacy of antileishmanial activity of drugs as compared to any other parasitic disease mainly due to the fact that Leishmania parasite resides within the macrophages which are responsible for clearance of liposomes in vivo [38]. Liposomal formulation with drug has been proved to be successful against leishmaniasis. Moreover, the use of conventional liposomes with antileishmanial drugs has been proved to be associated with the reduction in their toxicity profile [37].In fact macrophage surface contains receptors that recognize terminal galactose, mannose, fucose, or glucose residues of glycosides therefore sugar bearing liposomes were designed for improvement in macrophage targeting of antileishmanial agents [39]. In addition, mannose-grafted liposomal form was more efficient in transporting the drug to macrophages [40]. Furthermore, macrophages upon interaction with particulate drug delivery vehicles may act as secondary drug repository and contribute in localized delivery of the drug at the infected site [39].In addition, polymeric particles like synthetic aliphatic polyesters (polylactic acid) PLA, polyglycolic acid, and their copolymers (PLGA, or polycaprolactone) are the primary candidates for the development of nanoparticle-based delivery system. They offer several advantages as compared with liposomes: high drug-loading capacity, long-term stability, and suitability for oral administration. Moreover, they can control the drug release. When prepared with biodegradable and biocompatible polymers, they are welltolerated [41]. They may consist of either a polymeric matrix (nano- or microspheres) or of a reservoir system (nano- or microcapsules).In this regard, Venier-Julienne et al. used PLGA-NP for delivery of amphotericin B againstL. donovaniin vitro [18]. The activity of pentamidine loaded poly (D, L-lactide) nanoparticles against L. infantum in a murine model has been investigated by Durand et al. [19]. The activity and ultrastructural localization of primaquine-loaded poly (D, L-lactide) nanoparticles in L. donovani infected mice has been conducted by Rodrigues et al. [20]. The antileishmanial activities of 2′,6′-dihydroxy-4′-methoxychalcone by entrapment in poly(D,L-lactide) nanoparticles has been investigated by Torres-Santos et al. [21]. Durand et al. studied the activity of pentamidine loaded methacrylate nanoparticles against  L.infantum in a mouse model [22]. Gaspar et al. studied in vitro activity of primaquine-loaded poly(alkyl cyanoacrylate) nanoparticles against intracellular L.donovani [23]. Espuelas et al. studied in vitro antileishmanial activity of amphotericin B loaded in poly(epsilon-caprolactone) nanospheres [24].These nanoparticles in this study were prepared in the Department of Pharmaceutical  Biotechnology  and  Pharmaceutical  Sciences  Research  Centre,  Faculty  of  Pharmacy, Tehran  University of medical sciences. The antimony NPs in our study were composed of sulfur and antimony atoms at ratio of 84/16 and these particles were as Sb2S5 in their cytoplasm or other internal bacterial spaces according to Bahrami et al. Other characteristics of antimony sulfide NPs in this study and its green synthetic method are present in Bahrami et al. literature [28]. In our study, instead of using any additional compound for delivery of antimony sulfide, nanoparticle form of the drug was synthesized and its effects on Leishmania infantumin vitro condition were evaluated. Our results indicated the positive effectiveness of antimony sulfide  NPs  on  proliferation  of promastigote form. In addition, the drug can induce apoptosis in promastigotes. So these particles can be useful for elimination  of  parasite.  Surely this study was performed as preliminary work and further studies on the drug are needed.



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*Source: 756568-2012-06-26.xml*