In Vitro and In Vivo Trypanocidal Activity of H2bdtc-Loaded Solid Lipid Nanoparticles

The parasite Trypanosoma cruzi causes Chagas disease, which remains a serious public health concern and continues to victimize thousands of people, primarily in the poorest regions of Latin America. In the search for new therapeutic drugs against T. cruzi, here we have evaluated both the in vitro and the in vivo activity of 5-hydroxy-3-methyl-5-phenyl-pyrazoline-1-(S-benzyl dithiocarbazate) (H2bdtc) as a free compound or encapsulated into solid lipid nanoparticles (SLN); we compared the results with those achieved by using the currently employed drug, benznidazole. H2bdtc encapsulated into solid lipid nanoparticles (a) effectively reduced parasitemia in mice at concentrations 100 times lower than that normally employed for benznidazole (clinically applied at a concentration of 400 µmol kg−1 day−1); (b) diminished inflammation and lesions of the liver and heart; and (c) resulted in 100% survival of mice infected with T. cruzi. Therefore, H2bdtc is a potent trypanocidal agent.


Introduction
T. cruzi parasites are transmitted by insect vectors (triatomine bugs). T.cruzi is the causative agent of Chagas disease, which is silent and can remain asymptomatic for years [1], [2], [3]. A century after its discovery, this disease remains a serious public health issue-it is closely associated with human poverty and political instability as well as with little investment in drug development. According to the World Health Organization (WHO), between seven and eight million people are infected with T. cruzi worldwide, primarily in Latin America [4], [5], [6]. One in every four Chagas patients develops a fatal symptom of the disease due to lack of adequate diagnosis and treatment.
Nifurtimox and benznidazole (BZN) are currently available to treat the disease [7], [8], [9], [10]. However, neurological side effects have led commercial nifurtimox production to be discontinued [11]. As for BZN, although it is mainly effective during the acute phase of the infection, it presents undesirable side effects such as rash and gastrointestinal symptoms [12], so patients often fail to comply with the treatment [8]. Long treatment periods (30, 60, or 90 days) and appropriate pediatric formulations not available (administration of the medication to children often requires tablet fractionation) also limit BZN use [9], [11], [13]. A further concern is that no effective treatment for the symptomatic chronic phase of Chagas disease exists, so the patients usually receive palliative drugs at this stage [14], [15]. Therefore, a number of researchers are making considerable efforts to find new drugs to combat this disease.
Drug delivery systems can help to circumvent this problem. Because lipids have excellent physiological acceptability and can promote drug absorption as well as selective lymphatic uptake, researchers have focused on lipid-based drug release systems [23]. In particular, solid lipids constitute solid lipid nanoparticles (SLNs) at room and body temperature. Since SLNs consist of biocompatible and biodegradable lipids with low or no human toxicity, they can function as drug delivery systems [24], [25]. SLNs offer many advantages: they protect the drug against degradation, enable controlled drug release, and dismiss the use of organic solvents. Moreover, SLNs can be produced on a large scale, meeting industrial requirements [24], [26].
Our group has used H 2 bdtc in vitro experiments involving the Tulahuen strain (group I) [21]. The resistances of this group and of the Y strain (group II) have been reported to be different, based on phosphatase activities in T. cruzi homogenates [27]. Tulahuen had an optimum phosphatase activity at pH 4.0 and the Y strain at pH 7.0 [27]. Also in chronic phase has been associated with T. cruzi II-restricted infections [28]. In this sense, evaluating the trypanocidal activity of H 2 bdtc against the Y strain could support the use of this compound as a new drug against T. cruzi. In addition, so far little attention has been paid to the use of SLNs to treat Chagas disease [29], [30]. Therefore, this work investigates the in vitro and in vivo trypanocidal activity of free H 2 bdtc and H 2 bdtc encapsulated into SLNs (H 2 bdtc-SLNs) against the Y strain of T. cruzi and compares results with data obtained for the currently available drug BZN.
Particle size, dispersity index (D-stroke), and zeta potential determination The particle size and dispersity of the H 2 bdtc-SLNs dispersion were measured via photon correlation spectroscopy (PCS) [32]; the zeta potential was determined on the basis of the electrophoresis mobility of the nanoparticles using the Zetasizer ZS Nano 90 (Malvern Instruments, UK.). The samples were diluted (1:10) with distilled water at 25.0uC.

Atomic Force Microscopy (AFM)
The morphology of H 2 bdtc-SLNs was assessed using an atomic force microscope (ICON Bruker, USA). The samples were prepared by immersing freshly cleaved mica (Muscovite Mica Substrates Sheets, SPI Supplies, China) in SLNs aqueous dispersion and stored overnight, at room temperature, to complete the drying process. The samples were evaluated by AFM in the intermittent contact mode (tapping mode) by scanning the surface of mica (2 mm62 mm in area) using a rectangular silicon cantilever with a spring constant of 40 N m 21 vibrating at a frequency of 320 kHz. Imaging was performed at room temperature, and the topology image was used to determine the morphology of H 2 bdtc-SLNs [33].

Drug entrapment efficiency (EE%) determination
The total H 2 bdtc content in the H 2 bdtc-SLNs was determined by UV-vis spectroscopy at 400 nm (UV Spectrophotometer UV 1800, Shimadzu, Japan). First, a defined amount of H 2 bdtc-SLNs was dissolved in dimethyl sulfoxide. The amount of encapsulated drug was indirectly measured after centrifuging the H 2 bdtc-loaded SLNs for 40 min at 6000 rpm (1605 G), at 25uC in a centrifuge (Heraeus Megafuge 16 R Thermo Scientific, USA) equipped with a membrane concentrator (Amicon Ultra 15, MWCO 100 K, Millipore Corporation, USA). The filtrate was diluted with dimethyl sulfoxide (1:1), and the concentration of free H 2 bdtc in

Author Summary
The protozoan parasite Trypanosoma cruzi causes Chagas disease, a condition that affects the poorest regions of Latin America mainly. The chronic phase of this disease disables thousands of patients, constituting an important public health issue. The pharmacotherapy that is currently applied to treat the disease emerged many decades ago, is ineffective in most patients, mainly during the chronic phase, and has serious side effects. In a recent study, we showed that the compound 5-hydroxy-3-methyl-5-phenylpyrazoline-1-(S-benzyldithiocarbazate) (H 2 bdtc) is a potential drug candidate against the in vitro trypomastigote form of Tulahuen strains of T. cruzi. Here we report that H 2 bdtc loaded into solid lipid nanoparticles (H 2 bdtc-SLNs) displays good trypanocidal activity against the trypomastigote form of the Y strain of T. cruzi both in vitro and in vivo. Our in vivo experiments revealed that H 2 bdtc-SLN is 100 times more active than benznidazole (BZN), the drug that is commercially available to treat Chagas disease. Surprisingly, this compound has no side effects on the T. cruzi acute phase. Hence, we propose that H 2 bdtc-SLNs possesses interesting anti-Trypanosoma properties.
the diluted filtrate was determined using the same conditions employed to measure the total H 2 bdtc content used during the loading procedure (section 2.2). The amount of H 2 bdtc loaded into SLNs was calculated by subtracting the amount of free H 2 bdtc in the filtrate from the total amount of H 2 bdtc used during loading (26). EE (%) was determined using the following equation [26], [34].
EE%~d rug loading theoretical drug loading x100 Partition coefficient of H 2 bdtc (K octanol/water ) Partition coefficients for the H 2 bdtc were determined in triplicate in an n-octanol/water system following a published procedure [35]. Measurements of H 2 bdtc n-octanol/water partition coefficients were carried out using the shake-flask method. H 2 bdtc was dissolved in aqueous solution previously saturated with n-octanol at a concentration of 1 mg/mL and mixed with the same volume of octanol also previously saturated with water. Samples were stirred for 30 min, separate in two phases, and centrifugated for 10 min at 2000 rpm. The amount of H 2 bdtc in the aqueous phase was quantified by UV-visible spectroscopy.

Mice
Female Swiss mice (6 to 8 weeks old) were bred and maintained at the Department of Biochemistry and Immunology, School of Medicine of Ribeirao Preto, University of São Paulo, Ribeirão Preto, Brazil. The mice were maintained in microisolator cages under standard conditions; they were fed with food and water ad libitum.

Ethics statement
All the in vivo procedures were performed in accordance with the guidelines issued by the Brazilian College of Animal experimentation (COBEA) and received prior approval by the Ethics Committee on Animal Experimentation -CETEA (nu 006/2011) of the School of Medicine of Ribeirão Preto.''

Parasites and experimental infection
All the experiments were conducted using the trypomastigote form of the Y strain of T. cruzi (Lineage type II). For the in vitro experiments, parasites were grown in a fibroblast cell line (LLC-MK2). For the in vivo experiments, mice were intraperitoneally inoculated with 2.0610 3 bloodstream trypomastigote forms, which had been derived from previously infected Swiss mice.
In vitro evaluation of the trypanocidal activity and cytotoxicity of free H 2 bdtc and H 2 bdtc-loaded SLNs The trypanocidal activities of free H 2 bdtc, H 2 bdtc-SLNs, and BZN against the trypomastigote form of the T. cruzi Y strains were evaluated as described previously [36]. To this end, the trypomastigote culture at a concentration of 6.5610 6 parasites mL 21 was re-suspended in RPMI 1640 medium with 5% FBS. Triplicate cultures were treated with one of the investigated drugs and maintained at 37.060.1uC in a humidified atmosphere of 5% CO 2 . To test parasite viability, the number of motile forms was determined using a previously described method [37]. The concentration of compound corresponding to 50% trypanocidal activity after 24 h of incubation was expressed as the IC 50try (inhibitory concentration for the trypomastigote form).
Spleen cells isolated from C57BL/6 mice, macerated in RPMI 1640 medium (Gibco), and filtered using a 100-mm pore filter were used to evaluate the cytotoxicity in vitro. The isolated cells were centrifuged at 1500 rpm for 10 min, and erythrocytes were lysed in lysis buffer for 5 min, at room temperature. Cells were washed, counted, and resuspended at 6.5610 6 mL 21 in RPMI medium containing 5% fetal bovine serum. The spleen cells were seeded to a 96-well microplate (n = 2) and incubated for 24 h with H 2 bdtc diluted in dimethyl sulfoxide (DMSO, final H 2 bdtc concentration not exceeding 0.5%) or H 2 bdtc-SLNs (concentrations ranging from 125 mM to 0.24 mM in serial dilutions). BZN (Roche) was used as the reference drug; Tween 20 was employed as positive control for cell death. After the incubation period, the cells were washed and incubated with propidium iodide at a final concentration of 10 mg mL 21 . Cell cytotoxicity was measured on a flow cytometer (FACSCantoII -BD), and the data were analyzed using the FlowJo program (Tree Star).
In vivo evaluation of the cytotoxicity and trypanocidal activity of free H 2 bdtc and H 2 bdtc-SLNs Female Swiss mice aged between 6 and 8 weeks, weighing between 20 and 25 g, were infected with 2.0610 3 blood trypomastigotes per animal. A total of four experimental groups consisting of seven Swiss mice each were included in the study. Treatment started at day 5 post-inoculation (p.i.). BZN, free H 2 bdtc and H 2 bdtc-SLNs were orally administered at 4 mmol kg 21 (BZN 1.0 mg kg 21 day -1 /free H 2 bdtc and H 2 bdtc-SLNs 1.4 mg kg 21 day 21 ) per day for 10 consecutive days. The following treatments were applied: Group 1 = PBS control group; infected and not treated, Group 2 = infected and treated with BZN, Group 3 = infected and treated with free H 2 bdtc, and Group 4 = infected and treated with H 2 bdtc-SLNs. To evaluate parasitaemia and mortality, seven animals from each group were used. Seven animals were killed at day 22 p.i. (early mortality), to quantify inflammation of the heart and liver and to measure creatine kinase-MB (CK-MB) and glutamic-pyruvic transaminase (GPT) production.

Parasitemia and mortality
Parasitemia was analyzed on alternate days from day 7 p.i.; to this end, 5 mL of fresh blood was collected from the animal tail. The count of 100 fields was performed via direct observation under a light microscope [38]. Mortality was inspected on a daily basis until day 60.

Histological analysis
Groups of seven mice were euthanized at day 20 p.i., and portions of the heart and liver were fixed in paraffin for histological analysis. To assess inflammatory infiltration via light microscopy DP71 (Olympus Optical Co, Japan), tissues were sectioned at a 5-mm thickness and stained with hematoxylin-eosin (H&E). Each tissue section was imaged 25 times, and the percentage of the area occupied by cellular infiltrates was determined using the Image J program.

Serum activity of creatine kinase isoform MB (CK-MB) and glutamic-pyruvic transaminase (GPT)
The cardiac and hepatic lesions of mice infected with T. cruzi, treated or not, were assessed by measuring the creatine kinase-MB (CK-MB) and glutamic-pyruvic transaminase (GPT) levels, respectively, in the serum at day 22 p.i. The CK-MB levels were measured using a CK-MB kit (Liquiform, Brazil), as previously described [40]. Absorbance was measured on a microplate spectrophotometer (EMAX Molecular Devices Corporation, California, EUA). The color produced from this reaction was measured at a wavelength of 340 nm; the results are expressed in U/I. GPT was analyzed using an ALT/GPT kit (Liquiform, Brazil), according to the manufacturer's instructions. The colorimetric assay determines the amount of pyruvate produced according to the Reitman and Frankel method, from the formation of 2,4-dinitrophenylhydrazine [41]. The color produced by this reaction was measured at a wavelength of 505 nm.

Statistical analyses
Data are expressed as the mean SEM. Student's t-test was used to analyze the statistical significance of the variation between the infected and control assays. Differences were considered statistically significant when P,0.05. The differences in droplet size, dispersity, zeta potential, and entrapment efficiency values achieved during the stability test were evaluated via a one-way ANOVA analysis of variance followed by Tukey post-test analysis. The differences were considered statistically significant when P, 0.05. All the analyses were performed using PRISM 5.0 software (Graph Pad, San Diego, CA, US).

Preparation and characterization of H 2 bdtc-SLNs
The H 2 bdtc showed a lipophilic character (Log P (o/w) = 2.6960.03) and were efficiently encapsulated in this manner in SLNs. On the basis of Photon Correlation Spectroscopy (PCS), H 2 bdtc-SLNs had diameter of 127.4610.2 nm and dispersity lower than 0.3; the zeta potential revealed a negative surface charge (256.164.4 mV) ( Table 1). The entrapment efficiency was 98.1661.12, showing that the drug dispersed well within the lipid matrix. Atomic force microscopy images revealed that Table 1. Particle size, zeta potential, dispersity index and entrapment efficiency of SLNs (mean 6 SD, n = 3).

In vitro evaluation of the trypanocidal activity and cytotoxicity of free H 2 bdtc and H 2 bdtc-SLNs
We assessed the in vitro trypanocidal activity of free H 2 bdtc, H 2 bdtc-SLNs and BZN after 24 h of incubation with T. cruzi trypomastigotes forms. Free H 2 bdtc presents IC 50try (inhibitory concentrations against bloodstream trypomastigote) as 0.5060.12, H 2 bdtc-SLNs as 1.8360.18 and BZN 0.5060.39 mM ( Figure 3A). We also measured the cytotoxicity of free H 2 bdtc and H 2 bdtc-SLNs in spleen cells of Swiss mice; none of the tested drugs was significantly cytotoxic ( Figure 3B). Hence, both free H 2 bdtc and H 2 bdtc-SLNs displayed similar in vitro trypanocidal activity to BZN; this activity was not associated with general cytotoxicity but rather with specific activity against the parasite.

In vivo activity of free H 2 bdtc and H 2 bdtc-SLNs
We performed in vivo experiments to investigate the controlled release behavior of H 2 bdtc from H 2 bdtc-SLNs; we also compared the activities of free H 2 bdtc and H 2 bdtc-SLNs against T. cruzi. We decided to use an H 2 bdtc-SLNs concentration of 4 mmol kg 21 day 21 . During the in vivo treatments on the basis of preliminary in vivo results obtained for the Y strain of T. cruzi, which revealed that H 2 bdtc had low level of parasitemia (Supporting Information: Figure S1). In all the infected groups, parasitemia peaked at day 9 p.i., with gradual parasite elimination from the bloodstream after day 11 p.i. It is worth noting that we employed BZN concentrations 100 times lower than that used for Chagas patients. H 2 bdtc-SLNs eliminated 70% of the circulating parasites at the peak of infection, whereas free H 2 bdtc and the positive control BZN eliminated 48 and 15% of the parasites, respectively, as compared with the control group treated with PBS ( Figure 4A). In agreement with the data revealing reduced parasitemia, mice treated with H 2 bdtc-SLNs presented 100% survival rate ( Figure 4B), similar to the result achieved with BZN administered at a clinical dose of 400 mmol kg 21 day 21 (100 times more concentrated than the concentration used herein). Compared with the control group (PBS), groups treated with free H 2 bdtc and BZN exhibited a survival rate of 57%.

Cardiac and liver lesions
Encouraged by the in vivo results, we evaluated how free H 2 bdtc, H 2 bdtc-SLNs, and BZN affected the cardiac and hepatic tissues of the surviving animals. Infected mice treated with H 2 bdtc-SLNs presented reduced cardiac inflammation ( Figure 5A) and heart lesions were absent ( Figure 5B), as established by the absence of CK-MB, the enzyme released into plasma during cardiac lesion. Treatment with free H 2 bdtc diminished cardiac damage by 50% as compared with therapies with BZN or PBS. Concerning the ability of the tested compounds to reduce the liver damage caused by the parasite, H 2 bdtc-SLNs decreased inflammatory infiltration in the liver and hepatic toxicity more effectively, as assessed by measuring the glutamic-pyruvic transaminase (GTP) levels in the  serum ( Figure 5C, 5D). Considering all these results, it is possible to infer that treatment with H 2 bdtc per se reduced exacerbation of the inflammatory response on T. cruzi target organs and, consequently, tissue damage. Loading of H 2 bdtc into nanoparticles afforded even better results, producing no lesion in the heart tissue.
Because it is well established that parasites play an important role in cardiac damage during T. cruzi infection [42], [43], we quantified T. cruzi DNA derived from the heart tissues of mice treated with the tested compounds via real-time PCR. Treatment with H 2 bdtc-SLNs reduced the parasite burden significantly more effectively as compared with the other tested drugs (Figure 6), indicating that killing the parasites is most likely the mechanism through which H 2 bdtc-SLNs acts to diminish tissue lesions and enhance mice survival.

Discussion
Chagas disease has often been pointed out as being a major neglected disease; the drugs that are currently available to treat this disease are little effective [5], [12]. Efforts have been made to provide the affected populations with new compounds to treat the disease. In the past few years, researchers have tested many substances against T. cruzi. In particular, H 2 bdtc, which belongs to the class of S-dithiocarbazates, is efficient against the parasite [21]. A 24-h UV-vis study into the stability of H 2 bdtc in aqueous solution did not evidence any changes in the spectrum of this compound. Nevertheless, this drug is poorly soluble in water (1.50610 26 M), which has limited its use to treat Chagas disease. Because H 2 bdtc is lipophilic (Log P (o/w) = 2,6960,03) and SLNs constitute effective oral drug delivery systems, we loaded H 2 bdtc into this type of lipid.
We prepared the SLNs by the microemulsion method [31], [44], to avoid the use of organic solvents. The resulting SLNs had diameter of approximately 120 nm, dispersity lower than 0.3, and spherical shape, which made these lipids suitable for oral administration [45], [46], [47], [48]. The zeta potential measurement allowed us to predict the stability of the colloidal dispersion. Charged particles have high zeta potential-negative or positiveand usually do not aggregate [49]. The zeta potential results revealed that the SNPs prepared here had a negative surface charge (256.164.4 mV), indicating that the system was physically stable. Loaded and unloaded SNPs had similar zeta potentials, attesting that the tested drug was completely and uniformly dispersed inside the lipid matrix [50].
Encapsulation did not change the in vitro trypanocidal activity of H 2 bdtc, which was higher than the activity of BZN at the same concentration used here. The IC 50 values obtained for H 2 bdtc against the trypomastigote form of the Y strain of T. cruzi were comparable with or superior to those of previously reported active compounds [51], [52]. Other papers have also described the use of colloidal drug carriers such as liposomes and nanoparticles to treat Chagas diseases [53], [54]. Treatment of T. cruzi infection with a BZN-loaded liposome increased BZN levels in the liver and blood. Intravenous administration of free BZN and BZN-encapsulated liposome at 0.2 mg of BZN per kilogram of body weight revealed three-fold higher BZN accumulation in the liver in the second case. Nevertheless, encapsulation failed to improve the in vivo BZN efficacy [55].
Liposome instability prevents their use as drug delivery systems [56]. Fortunately, we verified that free H 2 bdtc and H 2 bdtc-SLNs were not toxic to the spleen cells of Swiss mice, which encouraged us to directly test the effect of H 2 bdtc formulations in vivo using a murine model of acute Chagas disease.
Treatment started with a relatively low oral dose of free H 2 bdtc and H 2 bdtc-SLNs (4 mmol kg 21 day 21 ) as compared with currently employed doses of the commercially available BZN and compounds tested in the literature [57], [58]. H 2 bdtc-SLNs, Free H 2 bdtc and BZN reduced the presence of parasites in the blood of infected mice in 70, 48 and 15% respectively. H2bdtc-SLNs maintained 100% survival rate of infected mice, whereas 43% of the mice treated with free H 2 bdtc or BZN at the same concentration succumbed to the disease. It is noteworthy that free SLN and PBS elicited similar levels of parasitemia (Supporting Information: Figure S2). Therefore, the use of SLNs as a drug delivery system increased the oral bioavailability of the target drug, as previously described [59], [60], [61], [62], [63]. H 2 bdtc loading into SLNs overcame the problems inherent to the poor water solubility of the compound and may be could make it more accessible to the parasite (however, detailed pharmacokinetic data will be presented in a separate forthcoming paper). Additionally, some authors have proposed that drugs loaded into SLNs measuring 20-500 nm are absorbed by lymphatic transport, which reduces the first-pass metabolism [62], [63].
Analysis of histological sections of liver and heart tissues (Supporting Information: Figure: Figure S3 and Figure S4) revealed that the inflammatory infiltrate decreased in all the treated groups as compared with the control. The reduction was more pronounced in mice treated with H 2 bdtc-SLNs, possibly because parasitemia was lower in this case. This corroborated with findings from previous studies [64], [65] and confirmed that the parasite elicited intense inflammation especially in the cardiac tissues. The fact that the heart tissues of mice treated with H 2 bdtc-SLNs were perfectly preserved agreed with the notion that the presence of inflammatory infiltrates is associated with cardiac tissue damage [66], [67] and also with parasitic load [42], [43]. Indeed, mice treated with H 2 bdtc-SLNs exhibited significantly lower parasite burden as compared with the other groups. Hence, the reduced parasitism elicited by H 2 bdtc-SLNs helps to preserve the heart tissues of mice infected with T. cruzi, allowing us to conclude that H 2 bdtc is a potent trypanocidal agent. Investigation into how H 2 bdtc interacts with possible targets represents a theme for future studies. For the time being, we must bear in mind that triazoles and thiosemicarbazones are well known for inhibiting cruzain, a protein belonging to the family of cysteine proteases and which is the most abundant protein in T. cruzi. Cruzain is essential for parasite development and survival within host cells [68]. H 2 bdtc bears pyrazole and dithiocarbazate parts, which are similar to triazoles and thiosemicarbazones, respectively, and could account for its trypanocidal action.
A mechanism of action similar to that of BZN probably does not occur. The BZN mode of action involves intracellular reduction of the nitro group, to produce highly reactive free radicals and/or electrophilic metabolites that could affect other systems, especially host systems, contributing to the cytotoxic effects observed in BZN-treated patients [69].
It is worth noting that cysteine proteases are very important for parasites; however, the lack of redundancy with respect to their mammalian hosts makes these proteases interesting targets for the development of new therapeutic agents [70]. Altogether, our findings show that H 2 bdtc-SLNs are a possible drug candidate to treat Chagas disease: it is more efficient against T. cruzi than the drugs used in current therapies. Figure S1 In vivo evaluations of the trypanocidal activity free-H 2 bdtc and H 2 bdtc-SLNs -(concentrations 4 mM, 40 mM and 80 mM) ( _____ ) Parasitaemia rate of mice infected with T. cruzi and treated with free-H 2 bdtc. (----) Parasitaemia rate of mice infected with T. cruzi and treated with H 2 bdtc encapsulated in solid lipid nanoparticles. Parasitaemia was monitored on days 7, 9, 11 and 13 after infection. The mean + SEM is shown and is representative of three independent experiments (n = 7). Statistically significant differences compared with the free-H 2 bdtc. T student test: **p,0.01 and ***p,0.001. (TIF) Figure S2 Parasitaemia of mice infected with T. cruzi and treated with free SLNs and PBS. Parasitaemia was monitored on days 7, 9, 11 and 13 after infection. (TIF) Figure S3 Cardiac lesions of T. cruzi-infected animals after treatment with H2bdtc encapsulated in SLNs. The sections represent of heart tissues inflammatory process composed of various cell types (21 days after infection). (TIF) Figure S4 Liver lesions of T. cruzi-infected animals after treatment with H 2 bdtc encapsulated in SLNs. The sections represent of liver tissues inflammatory process composed of various cell types (21 days after infection).