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. 2021 Apr 16;45(4):964–971. doi: 10.1007/s12639-021-01373-2

In vitro effect of artemether-loaded nanostructured lipid carrier (NLC) on Leishmania infantum

Meisam Khazaei 1, Vahid Rahnama 1, Mohammad Hossein Motazedian 1,2,✉, Soliman Mohammadi Samani 3, Gholamreza Hatam 1
PMCID: PMC8556446  PMID: 34789979

Abstract

Visceral leishmaniasis (VL) is an acute and deadly form of leishmaniasis, caused by Leishmania infantum parasite. Due to the toxicity and side effects of conventional treatment options, such as glucantime and other pentavalent drugs, finding novel drugs with fewer adverse effects is required. Artemether (ART), is one of the derivatives of artemisinin, which was shown to be effective in treating malaria and more recently, leishmaniasis. In this fundamental-applied research, we compared the effect of ART and nanostructure loaded with artemether (NLC-ART) on Leishmania infantum promastigotes and amastigotes, at different concentrations (2.5–5-10–25-50–100 μg/ml) using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method after 24 and 48 h of treatment. Inhibitory concentration (IC50) values (μg/ml) of promastigote and amastigote of L. infantum to ART/ NLC-ART, after 48 h of treatment, were found to be 37.12 / 32.1 and 16.43 / 15.42, respectively. Moreover, we found that (NLC-ART), had the lowest cytotoxicity against the J774 macrophage cell line. Conclusion: The NLC-ART can be a good candidate for the treatment of visceral leishmaniasis.

Keywords: Leishmania infantum, Artemether, MTT assay, Nanostructured lipid carrier (NLC), Drug delivery

Introduction

Leishmaniasis is an important vector-borne parasite disease that approximately infects two million people globally every year. Amastigotes of this parasite inoculate in the mammal’s skin, especially humans, by phlebotomine sandflies (Sacks and Sher 2002; Organization 2010; Asgari et al. 2019). Leishmaniasis is clinically manifested in three forms; cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL) (Sacks and Sher 2002; Goto and Lindoso 2010). Leishmania infantum and Leishmania donovani are the two species that can cause VL (Mohebali et al. 2005). Visceral leishmaniasis is highly endemic in countries, such as India, Nepal, Sudan, and Brazil (Murray 2002; Poche et al. 2016). In Iran, approximately 15,000 new cases occur annually, with the highest incident rate among children below two years of age (Alborzi et al. 2006, 2007). The main hosts for this disease are stray dogs, causing various symptoms, including irregular fever, lymphadenopathy, lymphocytosis, and monocytosis (20%–25%) (Lukeš et al. 2007; John and Petri 2013; McGWire and Satoskar 2013). In humans, leishmaniasis symptoms are fever, cough, weight loss, and hepatosplenomegaly. In patients with immune deficiency, VL can become a life-threatening problem (Desjeux 1999). The death rate in untreated VL is about 75%–95% (John and Petri 2013). The gold standard for the diagnosis of leishmaniasis is using microscopic and observational methods to detect amastigotes in the splenic and bone marrow aspiration. Serologic antibody and antigen tests are also used for the diagnosing (Singh and Sivakumar 2003; Junior et al. 2018). Moreover, molecular methods such as PCR are used to detect leishmania parasite DNA in the specimens collected from old and weak scars (Costa et al. 2015). Generally, antimonial pentavalent drugs are used as the first line of treatment for leishmaniasis. However, the major problems associated with the use of these drugs are the development of drug resistance and various side effects such as renal toxicity, hypotension, pancreatitis, anemia, leukopenia, thrombocytopenia, reversible renal insufficiency, and cardiotoxicity. Amphotericin B and pentamidine are in the second line of treatment, but due to the main problems of high toxicity and cost of these drugs, they are not widely used (Burgess and Birchall 1972; Croft et al. 1987; Le Fichoux et al. 1998; Basselin et al. 2002; Sundar and Chatterjee 2006). Recently, it was shown that artemisinin, the anti-malarial herbaceous drug, and its derivatives such as, artemether, can be used for the treatment of leishmaniosis and other parasitic diseases, such as schistosomiasis (Shuhua et al. 2000; Xiao et al. 2000; Milbradt et al. 2009). The effects of artemisinin and its derivatives, including artemether, on treating VL and Leishmania infantum have been previously demonstrated (Sen et al. 2010; Dehkordi et al. 2013a). Artemether and other derivatives of artemisinin as a herbal drug are relatively safe drugs. They have no dangerous side effects and unwanted reactions as prescribed. Also, because of some advantages such as usability in pregnant women, low toxicity, in addition to that this drugs reaction quickly and eliminated quickly so have recently attracted the attention of researchers (McGready et al. 1998; Shuhua et al. 2000; Xiao et al. 2000; Milbradt et al. 2009).

In the present study, a nanostructured lipid carrier, loaded with artemether (NLC-ART), was evaluated in vitro for the treatment of visceral leishmaniasis.

Materials and methods

Parasite culture

L. infantum standard strain (Mcan/IR/07/Moheb/-gh) was obtained from Shiraz University of Medical Sciences, Department of Parasitology and Mycology. Promastigotes of the parasite were cultured in RPMI 1640 (Shelmax Co, China) enriched with FBS 15% (v/v) (fetal bovine serum), 100 IU/ml of penicillin, and 100 μg/ml of streptomycin, ( All from Gibco, USA) and then incubated in 24 °C–26 °C.

J774 cells culture

In this eleven-month study, we used murine macrophage cell, (J774), obtained from Shiraz University of Medical Sciences Department of Immunology.

The cells were cultured in DMEM (Sigma, USA) enriched with FBS 10% (fetal bovine serum, 100 IU/ml of penicillin, and 100 μg/ml of streptomycin) and then seeded in 24-well plates and incubated at 37 °C and 5% CO2. Cells were nurtured with DMEM 10% after 17 h.

Preparation of nanostructure loaded with artemether (NLC-ART)

NLC loaded with artemether (NLC-ART) was obtained from Shiraz University of Medical Sciences, Department of Pharmaceutics School of Pharmacy, which was prepared during an unpublished student research project (16,141–01-01–1396). NLC-ART prepared based on the study of Ghasemiyeh et al. (2017).

Promastigote in vitro cytotoxicity assay

Briefly, promastigotes in the late logarithmic phase were seeded in 96-well tissue culture plates (1×106 cell/ml -100 µl per well). Drugs serial dilutions were then added to the wells (100 µl per well) at a concentration range of 2.5–5-10–25-50–100 μg/ml. Plates were then incubated at 24 °C at 24 and 48 h. cytotoxicity and inhibitory of the drugs were evaluated, using an MTT assay. After 24 h, a filtered MTT solution of 5 mg/ml in RPMI1640 (10 µl/well) in the dark condition was added to each well and incubation at 24 °C for 4 h. To dissolve the formazan crystals, 100 µl dimethyl sulfoxide (DMSO) was added to the wells and shaken for 15 min. Then, we read the absorbance at 570 nm with Elisa reader (Esfandiari et al. 2019).

Cell viability and cytotoxicity were calculated using the Formula:

Cellviability(%)=ODTest-ODBlankODcontrol-ODBlank×100

Cytotoxicity (%) = 100- Viability (%) (Barazesh et al. 2018).

J774 macrophages in vitro cytotoxicity assay

24 macrophage wells were poured into sterile falcons and centrifuged at 1500 rpm for 5 min. The supernatant was then discarded and 14 cc DMEM enriched with 10% FBS and also 100U/ml penicillin, 100 µg/ml streptomycin was added to the precipitate, and was well shaken. Macrophages were seeded in 96-well tissue culture plates (1×106 cell/ml per well), and then incubated at 37 °C and 5% CO2 for 24 h. Drugs serial dilutions in different concentrations (2.5–5-10–25-50–100 μg/ml) were then added to the wells (100 µl per well). Plates were then incubated at 37 °C and 5% CO2 at 24 and 48 h. After 24 h for evaluation of parasite viability, a filtered MTT solution of 5 mg/ml in RPMI1640 (10 µl/well) in the dark condition was added to each well and incubated at 37 °C and 5% CO2 for 2 h. To dissolve the formazan crystals 100 µl dimethyl sulfoxide (DMSO) was added to the wells and shaken for 15 min. Then, we read the absorbance in 570 nm with Elisa reader.

Cell viability and cytotoxicity were calculated using the.

formula:

cellviability(%)=ODTest-ODBlankODcontrol-ODBlank×100

Cytotoxicity (%) = 100- Viability (%) (Barazesh et al. 2018).

Statistical analysis

Graph Pad Prism 6 Demo was used for the statistical analysis. P-value less of than 0.05 was considered to be statistically significant. Data were analyzed using the ANOVA test (Barazesh et al. 2018).

Results

Anti-leishmanial activity of the formulations against promastigotes and amastigotes

Using MTT assay, we measured the viability of Leishmania infantum promastigotes in the presence of drugs after 24 h and 48 h of treatment at different concentrations (2.5–5-10–25-50–100 μg ml). As shown in Figs1 and 2, NLC-ART had the greatest effect on promastigotes at a concentration of 50 and 100 μg/ml during 24 h and 48 h of treatment. The P values for all concentrations at 24 h and 48 h are shown in Table 1, MTT test results on drug-treated Leishmania infantum promastigotes after 24 h indicated a significant difference between NLC-ART, and ART (P < 0.05). Moreover, the difference remained significant after 48 h, shown in Table 1 and 2.

Fig. 1.

Fig. 1

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Viability of the L. infantum promastigotes after 24 h at different concentrations of the formulations

Fig. 2.

Fig. 2

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Viability of the L. infantum promastigotes after 48 h at different concentrations of the formulations

Table 1.

P values for the viability of the L. infantum promastigotes after 24 and 48 h at different concentrations. Values are statistically significant at P < 0.05 as calculated by ANOVA test

C/ μg mL aART vs. NLC ART versus bNLC ART ART versus cAMB NLC versus NLC-ART NLC versus AMB NLC-ART versus AMB
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
2.5 0.6144 0.0093 0.0056  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001 0.0004  < 0.0001
5 0.2103  < 0.0001 0.0005  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001
10  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001
25  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001
50  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001
100  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001
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aArtemether vs. Nanostructured lipid carrier

bNanostructured lipid carrier loaded with Artemether

cAmphotericin B

Table 2.

P values for the macrophages after 24 and 48 h at different concentrations of the formulations. Values are statistically significant at P < 0.05 as calculated by ANOVA test

C/ μg mL aART vs. NLC ART vs.b NLC-ART ART vs.c AMB NLC versus NLC-ART NLC versus AMB NLC-ART versus AMB
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
2.5 0.9490 0.8561 0.9490 0.9208 0.0384 0.0675 0.9999 0.9985 0.0093 0.0088 0.0093 0.0138
5 0.4310 0.5986 0.6817 0.7556 0.0012  < 0.0001 0.9766 0.9938  < 0.0001  < 0.0001  < 0.0001  < 0.0001
10 0.0652 0.0675 0.3294 0.7556  < 0.0001  < 0.0001 0.8367 0.9153  < 0.0001  < 0.0001  < 0.0001  < 0.0001
25 0.0004  < 0.0001 0.0163  < 0.0001  < 0.0001  < 0.0001 0.6129 0.9862  < 0.0001  < 0.0001  < 0.0001  < 0.0001
50  < 0.0001  < 0.0001 0.0103  < 0.0001  < 0.0001  < 0.0001 0.3018 0.5179  < 0.0001  < 0.0001  < 0.0001  < 0.0001
100  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001  < 0.0001 0.2132 0.0285  < 0.0001  < 0.0001  < 0.0001  < 0.0001
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aArtemether versus Nanostructured lipid carrier

bNanostructured lipid carrier loaded with Artemether

cAmphotericin B

Half-maximal inhibitory concentration (IC50) values of promastigotes and amastigotes to all drug formulations are summarized in Table 3. IC50 values of promastigotes/amastigotes for NLC-ART, after 24 h and 48 h treatment, were obtained as, 31.52/16.43 and 27.95/15.42 μg/ml, respectively.

Anti-leishmanial activity of the formulations against amastigotes

Next, the anti-leishmanial activity of the formulations on J774 macrophage cell line infected with leishmania promastigotes was as assessed. After 48 h of culturing the infected J774 macrophage cells in the presence of drugs at different concentrations, macrophage viability and IC50 of amastigotes were determined (Figs. 5 and 6).

Fig. 5.

Fig. 5

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Evaluation of cytotoxicity of various formulations on J774 cells after 24 h. Amphotericin B is positive control

Fig. 6.

Fig. 6

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Evaluation of cytotoxicity of various formulations on J774 cells after 48 h. Amphotericin B is positive control

While NLC-ART showed negligible toxicity on J774 macrophage cells (Figs. 5 and 6), it was more effective in targeting the amastigotes residing inside the J774 macrophage cells as compared to ART.IC50 (μg/ml) for the NLC-ART: 27.95 (24 h) and 15.42 (48 h); for the ART: 45.2 (24 h) and 32.1 (48 h).

IC50, CC50, and selectivity index (SI) of promastigotes and amastigotes and all formulations are reported in Table 3.

Table 3.

IC50 (μg/ml), CC50 (μg/ml) and selectivity index (SI) (μg/ml) for promastigotes and amastigotes

Time/h ART NLC NLC-ART AMB
Promastigote IC50*
 24 57.73 2855 31.52 8.71
 48 37.12 1952 16.43 5.631
CC50**
 48 363.4 4853 788.7 86.51
Amastigote IC50
 24 45.2 1151 27.95 8.43
 48 32.1 1724 15.42 7.36
SI***
 48 9.789871 2.486168 48.00365 15.36317
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*Concentration of particles which has 50% inhibitory in promastigote forms

**Concentration of particles which has 50% cytotoxicity in macrophages

***Selectivity index

Determination of toxicity of formulations on J774 macrophage cells

To investigate the toxicity effect of formulations on J774 macrophage cells, we used the MTT assay. NLC-ART compared with ART and AMB it has the lowest toxicity significantly (Figs. 5 and 6).

Evaluating parasitic burden upon infection in J774A.1 cells

The parasitic burden after 24 and 48 h for infected J774 macrophage cells in the presence of drugs at different concentrations were determined and reported in Figs. 3 and 4, respectively. The data indicates that the NLC-ART, has less toxicity on macrophage cells than ART and AMB (Amphotericin B is positive control) Figs. 5 and 6.

Fig. 3.

Fig. 3

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Parasite burden after 24 h at different concentrations of the formulations

Fig. 4.

Fig. 4

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Parasite burden after 48 h at different concentrations of the formulations

Discussion

Visceral leishmaniasis is the most serious form of leishmaniasis disease that can be life-threatening if not treated. Currently, antimony pentavalent, amphotericin B, and pentamidine are among the drugs used for the treatment of VL. However, drug resistance and dangerous side effects are the major unsolved issues associated with these drugs. Therefore, discovering new drugs and/or new methods of drug delivery with low side effects and high therapeutic potential is essential (Santos et al. 2008).

The traditional antileishmanial drugs suffer from the long duration of treatment, difficulty of administration and low tolerability. In facing these challenges, nanotechnology can open a new avenue of therapies (Benita 2005).With the help of nanotechnology, drugs can be loaded onto well-organized nano carrier systems and be delivered to the site of interest. These nano carrier systems protect the drug from being metabolized, increase the bioavailability, and reduce the toxicity of drugs (Bobo et al. 2016).

Artemether has recently been used for the treatment of Leishmania and other parasitic diseases. With its low toxicity and good efficacy, artemether could be a suitable candidate for the treatment of leishmaniasis. Artemether induces cell death following activation in the presence of iron and production of free radicals (Bobo et al. 2016).

In recent years, several studies have addressed the effect of artemisinin and its derivatives on leishmaniasis. In an in-vivo study on BALB/c mice infected with Leishmania infantum, Sen, R. et al. described a significant reduction of parasite burden and splenic weight loss following treatment (Sen et al. 2010).

An in-vitro study reported that artemisinin was effective in eliminating the Leishmania major parasite through apoptosis induction of promastigotes (Ghaffarifar et al. 2015). Antileishmanial activity and toxicity of artemether were also studied by Ebrahimisadr, P.et al. In an in-vitro study. They showed that the artemether had the ability to inhibit the growth of intracellular and extracellular residing Leishmania major (Ebrahimisadr et al. 2013). The same group also studied the effect of artemether ointment on BALB/c mice lesions following infection with Leishmania. The result showed that the artemether significantly decreased the diameter of the lesions (Ebrahimisadr et al. 2013). The effect of artemether administration was evaluated on infected mice by the L.infantum parasite. And, accordingly the parasite burden decreased in the liver and spleen following oral treatment (Dehkordi et al. 2013b). In a toxicity study on citral treated mice, the nanostructured lipid carrier, encapsulated with citral (NLC-citral), it was found that the NLC-citral has a slow release function that this feature increases the solubility of citral in the water. Also, by blood biochemistry analysis, flow-cytometry immunophenotyping assay, and lipid peroxidation (NO), no toxicity was observed (Nordin et al. 2018).

The current study investigated the effect of a nanostructured lipid carrier loaded with artemether (NLC-ART) on treating the visceral leishmaniasis. The elimination of amastigotes within J774 macrophage cells showed that the NLC-ART drug was able to pass through the J774 macrophage cells membrane barrier; and doing so more effectively than free ART.

We are currently focusing on the in-vivo evaluation of NLC encapsulated drug in treating the visceral form of the leishmaniasis. Based on our findings presented in this study and our future in-vivo assessment of the drug, we are hoping to introduce NLC-ART as a potent drug to be used in clinical trials for treating this disease.

Conclusion

This study took the advantage of using nanotechnology in producing a new form of antileishmanial drug artemether. Nanostructured lipid carriers loaded with artemether were found to be more effective in eliminating the amastigotes and promastigote infected J774 macrophage cells. Importantly, the NLC and NLC-ATR had much lower toxicity than amphotericin B (as a positive control), and exhibited no toxicity to macrophage cells.

By examining the level of CC50 for J774 macrophage cells infected with amastigotes, we concluded that our drug passed from the cellular barriers freely, entered inside the infected J774 macrophage cells, and showed a promising effect eliminating the infection.

Acknowledgements

The results presented in this research were extracted from Meisam khazaei M.Sc. student. The dissertation was supported by the research council of Shiraz University of Medical Sciences.

Abbreviations

FBS

Fetal bovine serum

NLC

Nanostructured lipid carrier

NLC-ART

Artemether-loaded nanostructured lipid carrier

DMSO

Dimethyl sulfoxide; AMB: Amphotericin B

Author contributions

All authors contributed in this study: Parasite and cell culture, statistical analysis, promastigote in vitro cytotoxicity assay, J774 macrophages in vitro cytotoxicity assay, data collection, [Meisam Khazaei, Vahid Rahnama, Mohammad Hossein Motazedian, Soliman Mohammadi Samani, Gholamreza Hatam], preparation of nano structure loaded with artemether (NLC-ART) [Vahid Rahnama].

Funding

This study was financially supported by the vice-chancellor for research affairs of Shiraz University of Medical Sciences, Shiraz, Iran (Grant No. 97–01-01–17263).

Availability of data and materials

The dataset used and/or analyzed during the current study is available from the corresponding author upon reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

This study was approved by the ethics committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1397.1038).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The dataset used and/or analyzed during the current study is available from the corresponding author upon reasonable request.

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