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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.35 n.1 Santiago  2002 

Cytotoxicity and trypanocidal activity of nifurtimox
encapsulated in ethylcyanoacrylate nanoparticles


1. Programa Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile
2. Departamento de Farmacología, Facultad de Medicina, Pontificia Universidad Católica de Chile
3. Unidad de Parasitología Norte y Programa Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile
4. Departamento de Patología, Facultad de Odontología, Universidad de Chile


The aim of the present study was to study the trypanocidal activity of nanoparticles loaded with nifurtimox in comparison with the free drug against Trypanosoma cruzi, responsible for Chagas' disease. Ethylcyanoacrylate nanoparticles acted as the delivery system into cells. As the obligate replicative intracellular form is amastigote, in vitro studies were performed on this form of parasite as well as on cell culture derived trypomastigotes. The fluorescence method used here was very useful as it allowed for the simultaneous study of trypanocide activity and cytotoxicity by determining living or dead parasites within living or dead host cells. According to these results, the greatest trypanocide activity on cell culture-derived trypomastigotes was recorded for nifurtimox-loaded nanoparticles with a 50% inhibitory concentration (IC50) twenty times less than that of the free drug. The cytotoxycity of unloaded nanoparticles at low concentrations was similar to that obtained by free drug when evaluated on Vero cells. Furthermore, nifurtimox-loaded nanoparticles showed increased trypanocide activity on intracellular amastigotes with an IC50 thirteen times less than that of nifurtimox. We also observed that the unloaded nanoparticles possess the previously-described trypanocide activity, similar to the standard solution of nifurtimox, although the mechanism for this has not yet been elucidated. In conclusion, it was possible to establish in vitro conditions using nifurtimox encapsulated nanoparticles in order to decrease the doses of the drug and thus to obtain high trypanocidal activity on both free trypomastigotes and intracellular amastigotes with low cytotoxicity for the host cell.

Key terms: nifurtimox, nanoparticles, amastigotes, trypomastigotes, Trypanosoma cruzi.

Corresponding Autor: Gittith Sánchez. Programa de Biología Celular y Molecular, ICBM. Facultad de Medicina. Universidad de Chile. Independencia 1027. Casilla 70086. Santiago 7. Chile. Telephone: (56-2) 678-6757. Fax: (56-2) 735-5580 . e-mail:

Received: June 29, 2001. Accepted: December 28, 2001


The World Health Organization declared that Chagas' disease constitutes a serious health problem in more than fifteen countries. Twenty million people have already been infected, while another 90 million are at risk of infection (13). Trypanosoma cruzi, the causative agent, posses a high grade of genetic variability (18,20) and it passes through an obligatory multiplication phase in a form known as amastigote inside its vertebrate host cell. No ideal drug is available, and nifurtimox and benznidazol both have side effects that

limit their use, especially in adults (10). It is therefore important to be able to improve less toxic and more efficient protocols (1,11,12). The Increased activity of encapsulated drugs in polyalkylcyanoacrilate nanoparticles has been described previously (6). These authors reported that nanoparticles with fluorquinolons, such as pefloxacin mesilate and afloxacin, registered a minimal inhibitory concentration (MIC) to some positive and negative Gram bacteria that was 2 to 50 times less than that of the free drug. They also found better bioavailability, control of delivery time in blood. Moreover, Youseff et al (22) showed that ampicillin encapsulated in polyalkylcyanoacrilates nanoparticles has increased therapeutic activity in the treatment of Listeria monocytogenes, obtaining the same effect using doses of the antibiotic that are 20 times lower.

It is postulated that the absorption of nifurtimox in biodegradable nanoparticles of polyakylcyanoacrylate allows the delivery of the drug into the cell that hosts the parasite, thus increasing the trypanocide potency and lowering the toxicity by reducing the dosage (5). Previous in vitro studies on epimastigotes revealed considerably increased trypanocyde activity compared with that of a standard solution of nifurtimox. Furthermore, studies on cell cultures previously infected with metacyclic forms showed parasitism to be reduced by 87% - 94% whether the nanoparticles were loaded or unloaded with nifurtimox (7).

The aim of this study was to evaluate the in vitro trypanocidal activity of nifurtimox containing nanoparticles on cell-culture-derived trypomastigotes and on intracellular amastigotes. The fluorescence method used made it possible to simultaneously record the trypanocidal activity on parasites and cytotoxicity on host cell, in order to determine the best concentration to reach high trypanocidal activity with low cytotoxicity for the host cell.


I. Preparation of nanoparticles

Polyethylcyanoacrylates nanoparticles were prepared by polymerization according to the method described by Couvreur et al (3). The desired amount of nifurtimox (1 mg ml-1, solubilized with a minimal volume of dimethylsulphoxide, was dissolved in 50 ml of aqueous solution of 0.01M HCl containing 0.20 ml of the non-ionic surfactant Tween-20. An amount of 0.5 ml ethylcyanoacrylate monomer was added by drops over a period of 10 min with mechanical stirring (1000 rpm). After the polymerization was complete (commonly 3 hours) the colloidal suspension was adjusted to pH 7.0 with 0.2 M NaOH. The unloaded amount of nifurtimox was separated by centrifugation (Sorvall Superspeed RC2-B, Sorvall Inc, Newtown, CT). Finally, the nanoparticles were suspended in water and centrifugated again to exclude drug residue from the interparticle space.

II. Nifurtimox assay

The amount of nifurtimox bound to the nanoparticles was determined by a UV method. Normally, 40 mg of dried nanoparticles loaded with nifurtimox were dissolved in 12 ml of acetone. The organic solution underwent UV analysis. The UV apparatus consisted of a Milton Roy Spectronic 3000 spectrophotometer at 401 nm with a linear response in the range 3 to 12 ug ml-1. No interference was observed from the other components present in the nanoparticles (polyethylcyanoacrylate, Tween 20 and dymethylsulphoxide).

III. Morphological characterization and size analysis

Nanoparticle size was determined using a flow cytometer (FACS flow, Becton and Dickinson) fitted with an Enterprise 160 mW coherent laser at 488 nm; 60 mW at 535 nm. Data was collected from 10,000 particles for each sample at a flow rate of approximately 500 particles per second. The reading was made at an outgoing power of 60 mW at 488 nm. The fluorescent was collected with a 530/30 BP bandpass filter (FI 1 FITC), with the signal connected in logarithmic mode with the PTM FI 1=555 V and analyzed using the LEASES II Program (Becton and Dickinson).

IV. Parasite

Tissue culture-derived trypomastigotes from Vero cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY).

Parasites were collected five days post-infection from the supernatant infection. The strain used originated from the MF isolate, obtained from a chronic case of Chagas' disease in the Metropolitan Region of Chile, and was classified as zimodeme 1 (23).

V. Antiproliferative study in trypomastigote

Twenty five microliters of unloaded nanoparticles, nanoparticles loaded with nifurtimox equivalent in concentrations to a standard solution of nifurtimox, the standard solution of nifurtimox, and a solution containing only culture medium and dimethylsulfoxide were placed in each well of column 12 of a 12 x 8 well microtiter plate (Nunc, InterMed, Denmark). All the remaining wells of the plate received 50 µl medium for an eight-fold serial dilution. Fifty ml of medium containing 106 cell ml-1 was added to all wells for a final concentration of 5 x 105 cell ml-1. Medium only (50 µl) was added to the wells of the remaining rows, which acted as additional cell-free controls. The microtiter plates were placed in an incubator at 28ºC in a 5% CO2 atmosphere for 72 h. To prevent evaporation, plates were kept in a humidity chamber in the incubator. Parasites were counted in a Neubauer chamber (Boeco, Germany) under a microscope (Cambridge Instruments, Galen IL).

VI. Calculations of activity

T. cruzi growth inhibition was calculated using the equation:

% of activity = Nb - Nw

Where: Nb is a number of live parasites in the control and Nw is the number of parasites in each well.

VI Nanoparticles cytotoxicity assay

Vero cell line ATCC CCL-81 ase cultured at 37ºC in a moist atmosphere supplemented with 5% CO2 in RPMI-1640 medium with 10% fetal bovine serum previously inactivated at 56ºC for 30 min. The cells were washed twice and re-suspended in a complete RPMI-1640 medium. A 12 x 8 microtiter plate (Nunc, InterMed, Denmark) was used for the cytotoxicity assay. Each well received 100 µl containing 60 x 104 cells ml-1. After incubation at 37ºC for 12 hours in a 5% CO2 atmosphere, non-adhering cells were removed by rinsing with cold medium. After 24 hours of incubation with various concentrations of unloaded nanoparticles, nifurtimox-loaded nanoparticles, and standard solution of nifurtimox, cell viability was determined by the ionic-intensified fluorescein diacetate method (21). This method is based on the ability of the fluoresceindiacetate (FDA) to enter and diffuse into the cell. Viable cells emits a green fluorescence under UV light. Nonviable cells emitted red fluorescence of ethidium bromide. The viable cells were counted using a microscope with a UV light. The percentage of cytotoxicity was calculated by the formula:

  % Cd = 100 - % Cv (eq 2)
  % Cd = % non-viable cells
% Cv = % viable cell


VII. Trypanocidal activity on amastigotes forms

To study the trypanocidal activity of the nifurtimox-loaded nanoparticles in comparison with the nifurtimox standard, the same fluorescence method described above was used on Vero cells infected with amastigotes. According to the results obtained in the tests of tripanocidal activity and cytotoxicity on Vero cells, a narrower range of nanoparticle and nifurtimox concentrations was determined that shows a high tripanocidal activity and a low citotoxicity. Vero cells (4 x 104/well) were seeded and incubated for 24 hours, infected with 2 x 105 trypomastigotes per well for 4 hours. The cells were then washed three times with RPMI-10% fetal bovine serum and incubated for 24 hours. After that they were incubated for 4 hours with unloaded nanoparticles, nifurtimox-loaded nanoparticles or nifurtimox standard, washed and incubated for 24 hours, as in cytotoxicity assay. To quantify the tripanocidal activity, each well was observed under ultraviolet microscope and the dead amastigotes were counted using a control well as a reference.

VIII. Statistical test

The Student's t- test was used to compare the trypanocidal activity of the loaded and unloaded nanoparticles, as well as the standard of nifurtimox.


The nanoparticles had an average diameter of 195 ± 45 nm measured by flow cytometry. The amount of nifurtimox trapped in the nanoparticles was 8.2 ± 2.1 µg mg-1.

Table I shows the results of trypanocidal activity at eight different concentrations of nanoparticles and/or nifurtimox on free trypomastigote forms. Each value represents the mean value of five samples performed under the same conditions.

The highest trypanocidal activity was displayed by nifurtimox-loaded nanoparticles, reaching 100% with a concentration of 1.85 µg ml-1 of nifurtimox loaded nanoparticles. A three-fold concentration of the free drug (5.55 µg ml-1) is needed to obtain the same effect. Higher differences were observed at more diluted concentrations. With 0.21 µg ml-1 of the drugs, the loaded nanoparticles displayed an 83.1% activity level as compared with the 53.9% attained for free nifurtimox. At the lowest concentration studied, the mean percentage of activity obtained with nifurtimox loaded nanoparticles was 53.6% compared with 10.4 % of the free drug. Unloaded nanoparticles display trypanocide activity on the trypomastigote closeto that presented by standard nifurtimox. (Table I).

Trypanocidal activity (TA) on trypamastigote forms of unloaded nanoparticles (ULN),
nifurtimox-loaded nanoparticles (NLN) and standard solution of nifurtimox (SSN).

TA* (%)
TA* (%)
TA* (%)








*Mean value of five experiments.


The same effect can be observed by analyzing the values of the inhibitory concentration of the drug that lyse 50% of the trypomastigotes (IC50). Standard nifurtimox showed IC50 of 0.17 µg ml-1 compared with the concentration of 0.008 µg ml-1 of nifurtimox-loaded nanoparticles (Table II). These values represent an IC50 for nifurtimox loaded nanoparticles twenty times lower than standard nifurtimox.

Inhibitory concentration (IC50) on trypomastigote and amastigote forms of
nifurtimox-loaded nanoparticles (NLN) and standard solution of nifurtimox (SSN).



trypomastigotes 0.008 ± 0.003 0.17 ± 0.04
amastigotes 0.28 ± 0.06 3.86 ± 0.7

*Mean value of five experiments.


As can be observed in Table III, both unloaded nanoparticles and nifurtimox loaded nanoparticles displayed a cytotoxic effect on Vero cells. However at lower concentrations (0.21µg ml-1), the effect is comparable to that obtained for standard nifurtimox. At this concentration (Table I) the trypanocidal activity on trypomastigote forms of nanoparticles loaded with nifurtimox is higher (8.31%) than that obtained for standard solution of nifurtimox (53.9%).

Citotoxicity (CT) on vero cells of unloaded nanoparticles (ULN),
nifurtimox-loaded nanoparticles (NLN) and standard solution of nifurtimox (SSN)


3.8 ± 4.8
5.0 ± 3.0
4.0 ± 3.0
13.0 ± 6.7
3.8 ± 4.7
11.0 ± 5.4
16.0 ± 4.2
18.0 ± 8.3
12.5 ± 5.0
22.0 ± 4.5
26.0 ± 5.4
14.0 ± 5.4
56.0 ± 5.5
81.0 ± 11.4
30.0 ± 7.0
55.0 ± 5.0
86.0 ± 5.4

*Mean value of five experiments.


Table IV shows the trypanocidal activity on intracellular amastigotes of nifurtimox-loaded nanoparticles in comparison with unloaded nanoparticles and a standard solution of nifurtimox. The trypanolytic activity of nifurtimox loaded nanoparticles at concentrations of 1.59; 0.45 and 0.13µg ml-1 were 42%, 57.9% and 75.8% respectively, which corresponded to twice the trpanocidal activity displayed by the free drug. IC50 of nifurtimox loaded nanoparticles on amastigotes (0.28 µg ml-1) was 13 times lower than that for the standard solution (3.86 µg ml-1), as can be observed in Table II.


Trypanocidal activity (TA) on intracellular amastigotes of unloaded nanoparticles (ULN),
nifurtimox-loaded nanoparticles (NLN) and standard solution of nifurtimox (SSN).


23.4 ± 4.9 0.13 42.0 ± 5.7 21.2 ± 3.0
35.4 ± 8.0 0.45 57.9 ± 2.8 27.3 ± 6.4
46.4 ± 5.1 1.59 75.8 ± 7.1 35.6 ± 6.3

*Mean value of five experiments.



Previous studies conducted by our group (7) and others (3) have demonstrated the usefulness of polycyanoacrylate nanoparticles as lysosomatropic drugs. We have also recently demonstrated an increased in vitro trypanocidal activity of allopurinol-loaded nanoparticles (8). The findings shown in this and other studies with nanoparticles loaded with nifurtimox or allopurinol are highly relevant as no drug treatment has existed previously to cure the chronic phase of Chagas' disease. However, once the parasite enters the cell, drug treatments continue to be ineffective.

In our study the in vitro trypanocidal activity of the nifurtimox-loaded nanoparticles against T. cruzi was higher than a standard solution of nifurtimox. This fact could be explained by an interaction between the surface of the the nanoparticles and the outer part of the bilayers of the biological membranes, thus the surfactant coating of the nanoparticles surface may facilitate the penetration of the loaded drug into the parasite (17). On the other hand, Osuna et al (14,15) described a protein secreted by the metacyclic (infective) forms of the parasite capable of inducing the entry of inert particles into Hela cells. This permeabilizing protein induced endocytosis when the parasites interacted with Hela cells and facilitated the penetration of bentonita particles into the cells. These results suggest that the protein secreted by the parasite plays a key role in the penetration of this infective form into the host cell. This same mechanism may allow an enhancement of the entry of nifurtimox-loaded nanoparticles, thus increasing the usefulness of the carrier.

Our results showed that although high concentrations of unloaded and nifurtimox-loaded nanoparticles have greater cytotoxic effects on Vero cells than nifurtimox, it becomes similar to that of the free drug at more diluted concentratons. There is no explanation of this fact in the literature as most of the polymers used are polybutil and polyhexilcyanoacrylate whose degradation products are long chain alcohols that present low toxicity. The degradation of polymethylcyanoacrylate produces methanol, which is highly toxic and not used in these types of studies.

Unloaded nanoparticles presented trypanocidal activity similar to that presented by nifurtimox. Nevertheless, at low concentrations the higher activity of the former is considerable. At the most diluted concentration tested (0.01µg/ml) it displayed twice as much activity as nifurtimox. This fact was reported by Lherm et al (9), who performed in vivo assays of Trypanosoma brucei infected mice. The mechanism by which nanoparticles execute trypanocide action has not yet been elucidated. It is believed that enzymatic degradation would generate formaldehyde that would produce cellular lysis (4). In other reports, earlier degradation studies performed in vitro (2), have shown that the hydrolysis of the ester side chain is the primary route of degradation of polyalkylcyanoacrylates (PACA) nanoparticles. Furthermore, ester hydrolysis was shown to be catalyzed by enzymes of rat liver microsomes. Scherer et al (16) recently delineated the role of esterease on the degradation of polybutyl-2-cyanoacrylate (PBCN) nanoparticles. These authors found that the degradation rate of PBCN was proportional to the amount of esterase present, and in this way they confirmed the thesis formulated earlier (2) that the main route of degradation of PACA nanoparticles in biological condition is enzymatic. The probable metabolite products of PACA nanoparticles are polycyanoacrylic acid and the corresponding side chain alcohol.

The fact that IC50 of nifurtimox-loaded nanoparticles on the trypomastigote form is 20 fold lower than nifurtimox is of great relevance, since nanoparticles with lower doses of nifurtimox would be effective on extracellular trypomastigotes. The IC50 reported by González-Martins et al (7) of non-infective epimastigote forms of T. cruzi using the lowest concentration tested (0.008 mg/ml) was 50 times less than that of free nifurtimox, showing great differences between these two forms of the parasite. Also, nifurtimox-loaded nanoparticles showed an increased trypanocide activity on intracellular amastigotes, with an IC50 thirteen times less than for nifurtimox. This fact is highly relevant as nanoparticles with lower doses of nifurtimox can be used with the same efficacy.

As T. cruzi is an intracellular obligate parasite, it is very important to study the effect of nifurtimox-loaded nanoparticles on amastigotes within the host cell simultaneously with the evaluation of this cytotoxic activity. On intracellular amastigotes, unloaded nanoparticles presented trypanocidal activity similar to that displayed by nifurtimox; at low concentrations, both showed a similar cytotoxicity. In another intracellular parasite, Venier et al (19) reported the anti-leishmania activity of unloaded nanoparticles comparable to that of free drug amphotericin B.

The present study shows that nifurtimox-loaded nanoparticles clearly display a higher trypanocide activity on both trypomastigote and intracellular amastigote in comparison with unloaded nanoparticles and the nifurtimox standard. The in vitro-derived condition using lower doses of nifurtimox encapsulated in nanoparticles that exhibit low cytotoxicity stimulate in vivo experiments to determine the correspondence of the results in order to eliminate or decrease the adverse effects and increase the efficacy of the chemotherapeutic protocols used at present.



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