Encapsulation of benznidazole in nanostructured lipid carriers and increased trypanocidal activity in a resistant Trypanosoma cruzi strain

Abstract Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi, whose treatment has remained unsatisfactory for over 50 years, given that it is limited to two drugs. Benznidazole (BZN) is an efficient antichagasic drug used as the first choice, although its poor water-solubility, irregular oral absorption, low efficacy in the chronic phase, and various associated adverse effects are limiting factors for treatment. Incorporating drugs with such characteristics into nanostructured lipid carriers (NLC) is a promising alternative to overcome these limiting obstacles, enhancing drug efficacy and bioavailability while reducing toxicity. Therefore, this study proposed NLC-BZN formulations in different compositions prepared by hot-melt homogenization followed by ultrasound, and the optimized formulation was characterized by FTIR, DRX, DSC, and thermogravimetry. Biological activities included in vitro membrane toxicity (red blood cells), fibroblast cell cytotoxicity, and trypanocidal activity against epimastigotes of the Colombian strain of T. cruzi. The optimized NLC-BZN had a small size (110 nm), negative zeta potential (-18.0 mV), and high encapsulation (1.64% of drug loading), as shown by infrared spectroscopy, X-ray diffraction, and thermal analysis. The NLC-BZN also promoted lower in vitro membrane toxicity (<3% hemolysis), and 50% cytotoxic concentration (CC50) for NLC-BZN in L929 fibroblast cells (110.7 µg/mL) was twice the value as the free BZN (51.3 µg/mL). Our findings showed that the NLC-BZN had higher trypanocidal activity than free BZN against the epimastigotes of the resistant Colombian strain, and this novel NLC-BZN formulation proved to be a promising tool in treating Chagas disease and considered suitable for oral and parenteral administration.


INTRODUCTION
Chagas disease (CD) is an anthropozoonosis affecting millions of people; it is considered one of the most neglected tropical diseases and is estimated to be responsible for 12,000 deaths and 56,000 new cases yearly (WHO, 2020;PAHO, 2020).This condition is caused by the protozoan Trypanosoma cruzi and is characterized by two distinct clinical phases.The initial acute phase is associated with high parasitemia, lasting 4-8 weeks.The late chronic phase is characterized by the evolution of the disease to critical clinical manifestations in the heart or gastrointestinal tract, albeit with a remarkable decrease in parasitemia.Some patients may even have an indeterminate form of CD and remain asymptomatic along these phases (Prata, 2001).
The condition of a neglected disease is clearly observed in the history of CD therapy, which has remained the same since the 1970s; it is restricted to only two drugs: nifurtimox (NFX) and benznidazole (BZN) (Romero, Morilla, 2010).Despite both drugs having parasitological cure rates above 70-80% in the acute phase, they are not efficient in the chronic phase (Morilla, Romero, 2015).Moreover, both BZN and NFX cause various adverse effects, usually leading to treatment discontinuation by the patients.As BZN shows better tolerability, a broader action spectrum, and higher tissue penetration, it is usually preferred over NFX and considered the first-choice drug in CD treatment, although researchers have reported resistance of T. cruzi strains to BZN (Maximiano et al., 2010;Salomon, 2012;Perez-Molina, Molina, 2018).
Drug encapsulation in nanocarrier systems is an important strategy used to increase the solubility and bioavailability of many poorly water-soluble drugs, and incorporating BZN into lipid nanoparticles is a relevant field to be explored.Nanostructured lipid carriers (NLC) are the second generation of lipid nanoparticles, and the solid matrix consists of a mixture of solid and liquid lipids, where the drug molecules can be encapsulated.These nanocarriers have been successfully proposed to improve many therapies, such as cancer, heart and brain diseases, antibiotics, and even vaccines.The presence of oils makes the solid lipid matrix more unstructured and with more imperfections to accommodate higher quantities of the drug (Gaba et al., 2015;Beloqui et al., 2017).The high biocompatibility of the excipients in their composition is vital for safe drug use; at the same time, these nanosystems can provide other benefits for the BZN treatment, their high stability during storage, the potential for controlled drug release from the lipid matrix, enhanced oral absorption, and versatility in administration through different routes (Gaba et al., 2015;Beloqui et al., 2017).
Few efforts have been made to propose lipid nanocarriers for BZN incorporation in the form of liposomes (Morilla et al., 2002;Vinuesa et al., 2017), nanoemulsions (Streck et al., 2014), microemulsions (Streck et al., 2016), and even lipid nanoparticles (Vinuesa et al., 2017).However, the efficiency of BZN incorporation in all these systems is low in terms of drug/lipid ratio, and high encapsulation has only been reached with high lipid amounts.Given the limited and unsatisfactory CD therapies available and the potential of NLC for high incorporation of poorly water-soluble drugs, this study sought to develop BZN-loaded NLCs with higher drugloading capacity in low lipid amounts in order to address the cytotoxicity profile and in vitro trypanocidal activity of the optimized formulation.

Development of BZN lipid nanoparticles
As utilized in previous studies (Marcial, Carneiro, Leite, 2017;Fernandes et al., 2018), the composition of the NLC matrix consisted of Compritol and Crodamol GTCC (medium chain triglycerides, MCT) as solid and liquid lipids, respectively.Surfactants were proposed alone or mixed: Tween 80 (TW80), egg lecithin (EL), soy lecithin (SL), and poloxamer 407 (P407).Cholesterol (Chol) was utilized as a co-surfactant when present.The main goal at this stage was higher BZN incorporation with smaller and homogeneous particle sizes.

Nanoparticle preparation
Formulations were prepared by the hot-melt homogenization method followed by ultrasonication, as reported elsewhere (Marcial, Carneiro, Leite, 2017).Briefly, the oily phase (OP) containing lipids and surfactants was heated at 85 °C and the aqueous phase (AP), a 2.25% (w/v) glycerol solution in water, was preheated at the same temperature (batch: 20 mL).When present, BZN was previously solubilized in the OP.Next, AP was slowly poured onto the OP, and the mixture was stirred for 2 min and immediately homogenized with a high-intensity ultrasonic probe (Q55 sonicator; Qsonica, Church Hill Road, Newton, USA) for 10 min (40% amplitude).The final pH was adjusted to 7.0.

Particle size and zeta potential
The average hydrodynamic diameter and zeta potential (ZP) were determined in a Zetasizer Nano ZS (Malvern Instruments; Worcestershire, England) at a 173° angle and 25 ºC.Formulations were 10-fold diluted in AP before the measurements.All determinations were performed in triplicate, and the maximum acceptable value to consider the sample monodisperse was a polydispersity index (PDI) of 0.3.

Drug encapsulation efficiency
The BZN quantification was performed using the HPLC method previously developed and validated (Almeida et al., 2016).Encapsulation efficiency (EE) was determined from the quantification of BZN before (total BZN) and after filtration (filtered BZN) of the NLC dispersion in a 0.45-μm PVDF membrane (Millipore, Billerica, USA).The BZN concentration solubilized in the external aqueous phase was determined after 0.45 μm filtration and ultrafiltration (Amicon 100 k, Millipore, Billerica, USA) and was negligible.
Thus, 200 µL of the nanoparticle dispersion in both conditions (total and filtered) was dissolved in tetrahydrofuran (4 mL) and diluted to 5 mL in methanol to be analyzed by HPLC.The EE was then calculated using the equation:

Stability of the nanoparticle dispersion
The NLC-BZN formulations (n = 3) were stored at 4 °C, protected from light, and monitored as the average diameter, PDI, ZP, and pH at 0, 7, 14, and 28 days.BZN retention was also monitored considering the percentage of BZN kept encapsulated in the nanocarrier throughout the time (vs.time 0).

Freeze-drying conditions
The NLC-BZN and b-NLC water dispersion were frozen in liquid nitrogen for 5 min and lyophilized in a Labconco FreeZone 4.5-L freeze-dryer (Kansas, EUA) for 24 h at -50 ºC for further characterization.

Characterization of lyophilized NLC-BZN (L-NLC-BZN)
Fourier transform infrared spectroscopy (FTIR) FTIR absorption spectra were obtained from a Varian 640-IR spectrophotometer (Palo Alto, USA) equipped with attenuated total reflectance mode.Spectra were obtained for the formulation components in the physical state, the lyophilized b-NLC and NLC-BZN, and the physical mixture (b-NLC + BZN) at room temperature using a diamond crystal with 4 cm -1 resolution and 32 accumulations.
Powder X-ray diffraction PXRD data were collected in an XRD-7000 diffractometer (Shimadzu, Kyoto, Japan) at room temperature under 40 kV and 30 mA using CuKα (λ = 1.54056Å) equipped with polycapillary focusing optics under parallel geometry coupled with a graphite monochromator.The sample was spun at 60 rpm and scanned over an angular range of 2-40° (2θ) with a step size of 0.02° (2θ) and a time constant of 2s/step.All fitting procedures were obtained using FullProf Suite.
For the thin film experiment, conditions were room temperature under 40 kV and 35 mA using CuKα (λ = 1.54056Å) equipped with polycapillary focusing optics under parallel geometry coupled with a graphite monochromator, rising angle of 1°, in a static sample setup, and using 0.02° (2θ) as step increment and time constant of 4s/step.

Thermal analysis
Differential scanning calorimetry (DSC) and thermogravimetry (TG) analyses were performed for the solid formulation components (BZN, Compritol, and poloxamer 407), and the lyophilized formulations L-b-NLC and L-NLC-BZN.The DSC curves were obtained in a DSC60 Shimadzu cell (Tokyo, Japan) and calibrated with indium (T onset = 156.63°C, ΔH fus = 28.45J/g) under the following conditions: dynamic nitrogen atmosphere at 50 mL/min, a heating rate of 10 °C/min, from 30 to 400 °C, and sample mass of about 1.5 mg accurately weighed in a closed alumina crucible.The TG curves were obtained using a Shimadzu DTG60 thermobalance (Tokyo, Japan) with a heating rate of 10 °C/min, from 30 to 600 °C, dynamic nitrogen atmosphere of 50 mL/min, and sample mass of about 2.5 mg accurately weighed in an aluminum crucible.

In vitro membrane toxicity studies
In vitro membrane toxicity studies were performed according to Scalise et al. (2016), with few modifications.In summary, 100 μL of the 4% RBC suspension and 100 μL of the treatments (BZN solution, b-NLC, and NLC-BZN) were added to a 96-well plate in BZN concentrations of 12.5, 25, 50, and 100 μg/mL.The negative control was a saline solution (0.9% w/v), and the positive control was a 1% (w/v) saponin solution.The plates were then shaken for 1 h (400 rpm; 37 °C) and centrifuged (3000 rpm; 15 min).The supernatant was collected, and the amount of released hemoglobin was determined by UV spectrophotometry at 540 nm.The percentage release of hemoglobin was calculated using the equation:

Cell viability studies
In vitro cytotoxicity studies were conducted in L929 cells (mouse fibroblasts) grown in RPMI 1640 medium supplemented with 10% FBS, 3.2 mM L-glutamine, 100 µg/mL streptomycin, and 100 IU/mL penicillin in a humidified incubator with 5% CO 2 at 37 °C.The L929 cells (1.0 x 10 4 cells/well) were seeded in 96-well plates and incubated for 24 h.After this time, the treatments (free BZN solution and NLC-BZN) were added to the plates in the range of 1.95 to 125 μg/mL, and a 0.02 M cadmium chloride solution was utilized as a positive control.After 72 h of incubation, 20 μL of an MTT solution (3 mg/mL) was added to each well, and the microplates were incubated at 37 °C for 4 h.Afterward, the supernatant was discarded, and DMSO was added to dissolve the formazan crystals.The metabolic activity was estimated as the MTT conversion rate by measuring the absorbance at 540 nm (Mosmann, 1983).The absorbance of wells with non-treated cells was considered 100% cell viability, and the results from three independent experiments were expressed as the relative percentage of cell viability, and CC 50 was determined.

Trypanocidal activity
The trypanocidal activity of NLC-BZN was investigated using epimastigotes of the Colombian strain of T. cruzi, which is considered a prototype of resistance to BZN (Filardi, Brener, 1987).The strain was harvested in LIT medium supplemented with 10% FBS, 0.2% streptomycin solution (100 μg/mL), and 0.18% hemin solution (10 mg/mL).The culture was maintained in a biochemical oxygen demand (BOD) incubator at 26 ºC, and the experiments were performed after the strain reached the stationary phase.
The T. cruzi strain (2.0 x 10 6 parasite/well) was seeded into 96-well plates, and the treatments were added: free BZN solution and NLC-BZN, in the range of 1.95 to 250 μg/mL; b-NLC was diluted in the same way as NLC-BZN.The plates were incubated at 26 ºC in the BOD incubator for 72 h, then the viable parasites in the supernatant were counted in a Neubauer chamber (Bortoluzzi et al., 2021;Pereira et al., 2021).The number of parasites from the untreated wells was considered as 100% cell viability.Results from three independent experiments were expressed as the relative percentage of cell viability, and the 50% inhibitory concentration (IC 50 ) was also determined for the treatments.

Statistical analysis
The results were represented as mean value ± standard deviation from determinations in triplicate.Oneway analysis of variance (ANOVA) followed by Tukey's test was used to analyze the statistical differences among the mean values (α = 0.05).Data from cell viability were analyzed by two-way ANOVA followed by Bonferroni post-test (α = 0.05).

Formulation development
Despite the undeniable importance of BZN as an antichagasic drug, its properties are not favorable for oral administration: low aqueous solubility (0.4 mg/mL) and irregular absorption, resulting in unfavorable pharmacokinetics (Maximiano et al., 2011a).Incorporating these poorly water-soluble drugs into lipid nanoparticles can be an alternative for increasing drug bioavailability and stability in physiological conditions, including those found in the gastrointestinal tract, while comprising a stable and versatile product.Other lipid nanocarriers, such as liposomes and nanoemulsions, may be unstable in the gastrointestinal tract or during storage (Gaba et al., 2015;Beloqui et al., 2017).
Four parameters were varied in the optimization stage of the BZN-loaded NLC: the liquid lipids, the composition of the surfactant system, the total lipid/ surfactant ratio, and the amount of BZN initially added for encapsulation, according to the composition described in Table I.All formulations were screened and optimized to find the smallest size with homogenous distribution, higher ZP (in absolute values), and higher EE.Thus, the changes performed in the composition of liquid lipids and inclusion of Chol had negligible influence on the particle size and PDI, which remained near 115 nm and 0.25, respectively (p > 0.05).However, EE drastically reduced from 83.0% (NLC A) to 74.5% (NLC B) and 74.2% (NLC C), which was likely a result of the different BZN solubility in the lipid combinations (Table I).Thus, the MCT was chosen as the liquid lipid in the lipid matrix.
Nonetheless, in these initial formulations containing only TW80 as a surfactant, BZN was released only a few hours after the NLC preparation, clearly indicating poor encapsulation stability (50-60% release after 3-4 h).The surfactant system was then varied in mixtures of TW80 with P407, SL or EL, and P407 alone.NLC F (containing TW80 + P407) was the most promising, as it had a similar diameter, ZP, and EE of the initial NLC A (only TW80), albeit PDI decreased to 0.19 (Table I), indicating a more homogeneous size distribution and lack of initial release of BZN.Larger particle sizes and lower EE were observed in the other formulations: NLCs D, E, and G (when only P407 was utilized).The ZP was negative in all formulations, ranging from -15.9 to -46.0 mV, which may be associated with the presence of ionized free fatty acids at the interface of the particle, naturally present in the lipids and surfactants: MCT, soybean oil, lecithin, and Compritol (Bruxel et al., 2012).
To increase the BZN encapsulation, some changes in the total lipid/surfactant ratio were proposed.However, only increased particle size (up to 330 nm) was found in NLC H and I without increasing the EE (Table I).In fact, increased diameter is expected with a higher total lipid/ surfactant ratio, although the use of higher quantities of lipids is better justified with higher encapsulation, as the excessive increase in the lipid content is associated with higher cytotoxicity caused by the increase of free fatty acids resulting from the enzymatic degradation of the lipid matrix (Scholer et al., 2002;Ridolfi et al., 2011).
Therefore, the lipid matrix of NLC F was utilized in the final analyses.
Therefore, NLC F (with 0.05% BZN) was considered the optimized formulation.This product had 100% of the particles with an average diameter below 500 nm and 85.9% below 200 nm, meeting the particle size requirements for oral and even intravenous administration (USP39-NF34, 2016).The low mean diameter of this optimized NLC-BZN (110 nm), with a narrow size distribution (PI = 0.19), is also associated with higher cellular uptake and high absorption when compared with larger nanoparticles (Li et al., 2016;Beloqui et al., 2017;Danaei et al., 2018).
All analyzed parameters (diameter, PI, ZP, and pH) of the optimized NLC-BZN remained stable for up to 28 days (Figure 1).There was no change (p > 0.05) in the concentration of BZN retained in the matrix, and only a reduction of roughly 6.2% was detected after 28 days.The increase in the nanoparticle size usually precedes other macroscopic changes and is a good indicator of instability (Bahari, Hamishehkar, 2016).The negative ZP, together with the presence of ethoxylated surfactants (TW80 and P407), hinder the natural tendency of nanoparticle aggregation either by electrostatic repulsion or steric hindrance of the hydrophilic polyethylene oxide chains coating the nanoparticle surface (Wulff-Perez et al., 2012;Beloqui et al., 2017).

Fourier transform infrared spectroscopy
The FTIR absorption spectra for the pure materials of the NLC-BZN are shown in Figure 2A.All characteristic absorption bands were observed for the pure components, with the corresponding wavenumber (cm -1 ) and intensity summarized in Table II.The FTIR spectrum of b-NLC (Figure 2B) showed a full and robust band at 3400-3200 cm -1 (O-H from glycerol) and in the range of 2900-2700 cm -1 (C-H stretch from the various components).The absorption spectra of NLC-BZN and b-NLC had a similar profile, and the characteristic bands of BZN (3330, 1685, 1660-1500, and 1250-1000 cm -1 ) could only be observed in the spectrum of the physical mixture.Together with the high EE values, this may indicate BZN encapsulation inside the lipid matrix.

Powder X-ray diffraction
As shown in the diffractograms in Figure 3A, Compritol showed two diffraction peaks for NLC-BZN, a high-intensity peak at 21.2º 2θ and a lower intensity peak at 23.4º 2θ, with the characteristic values for the β' form of the lipids (Fini et al., 2011).Poloxamer 407 showed two characteristic high-intensity peaks at 19.4º and 23.5º 2θ, as described elsewhere (Garcia-Millan, Quintans-Carballo, Otero-Espinar, 2017).Furthermore, BZN presented a typical crystalline diffraction pattern, exhibiting sharp peaks at 11.0º, 16.9º, 21.9º, and 25.2º 2θ, a similar profile observed in previous reports (Palmeiro-Roldan et al., 2014).
In the experiment with the thin film (Figure 3A, solid thin line), NLC-BZN did not show the characteristic signal of BZN at about 20.5° 2θ, indicating that BZN is not present at the surface of the sample.Thus, during the nanoparticle preparation and in contact with the lipids of the matrix, BZN may have undergone drug amorphization or molecular solubilization, which could have contributed to encapsulating BZN in the lipid matrix, thereby corroborating the high EE values.

Thermal analysis
The DSC curve of BZN showed an endothermic event at 188.38 °C (ΔH = 133.01J/g), corresponding to the melting point (Soares-Sobrinho et al., 2010).An exothermic event starting at 220 °C, characteristic of drug decomposition (Figure 3B), was confirmed by the loss of mass in the same temperature on the TG curve (Figure 3C).The endothermic events of Compritol (T onset = 69.60 °C; ΔH = 117.76J/g) and Poloxamer 407 (T onset = 51.39 °C; ΔH = 117.48J/g) were also consistent with the melting point described (Fini et al., 2011).
The thermal behavior for both NLC-BZN and b-NLC formulations were similar, with a single endothermic event at 64.07 and 63.74 ºC (ΔH = 85.79 and 87.40 J/g), respectively (Figure 3B), without loss of mass in the TG curve, which was associated with the melting of the nanocarriers.The formulations presented thermal stability up to 238 ºC, a higher temperature than BZN alone.At this temperature, the decomposition process begins, with a 93% loss of mass in two stages (Figure 3C).
Therefore, the high EE obtained for BZN in the optimized NLC formulation was also observed in the FTIR, PXRD, DSC, and TG studies due to the similarities observed between the signals of the BZNloaded NLC and the blank formulation (without BZN, b-NLC).As observed in the other characterization studies, the missing signals of BZN in the NLC-BZN analyses are also indicators of this high encapsulation.The broad and partially symmetrical edges of the peaks found in the DSC curve of the b-NLC and NLC-BZN are typical of complex systems, such as multicomponent carriers.The observed decrease in the melting enthalpy of NLC compared to the pure Compritol is evidence of the lower crystallinity degree of the NLC matrix (Li et al., 2009;Gonullu et al., 2015;Argimón et al., 2016).Since none of the thermal events attributed to the NLC-BZN were exclusively assigned to any of the isolated raw materials, one can assume that a singular formulation with specific physicochemical characteristics was produced, and the selected proportion of each component was solely part of the nanoparticle composition (Bunjes, Unruh, 2007).

Membrane toxicity studies
The next step was investigating the in vitro toxicity of the developed BZN nanocarriers.The RBCs are an indicative model of membrane toxicity and a straightforward method since the hemolytic activity promotes hemoglobin release from RBC, which can be quantified by spectrophotometry (de Freitas et al., 2008).Percentage hemoglobin release was low (<9%), even at the highest concentration (100 μg/mL), regardless of the treatments applied (Figure 4A).Similar findings were previously obtained by Scalise et al. (2016) for BZN nanocrystals (6% hemolysis at 100 μg/mL) and conventional BZN (9% hemolysis at 50 μg/mL).
Moreover, the percentage of hemoglobin release was even lower (<3%) for nanocarrier treatment (b-NLC and NLC-BZN).At the highest concentration tested (100 μg/mL), there were no significant differences between the hemolysis caused by free or encapsulated BZN, indicating the low toxicity of NLC-BZN to membranes by direct contact.

Cell viability
The L929 cell viability (human fibroblasts) was dosedependent after 72 h of treatment (Figure 4B), and NLC-BZN was less cytotoxic for L929 cells than free BZN.For instance, the cytotoxicity of free BZN (16.8 ± 2.0%) was higher than NLC-BZN (56.5 ± 7.1%) at the concentration of 125 μg/mL, and a similar profile was observed at the other concentrations.The CC 50 determined for NLC-BZN (110.7 ± 1.1 μg/mL) was twice the value of free BZN (51.3 ± 1.1 μg/mL for BZN).
After 72 h, the treatment with free BZN was clearly more cytotoxic for L929 cells than the NLC-BZN, indicating favorable biocompatibility of the developed lipid nanoparticles.The toxicity of BZN in mammalian cells seems to be associated with non-specific and highly reactive electrophilic metabolites (Castro, de Mecca, Bartel, 2006), and its encapsulation should reduce cytotoxicity in normal cells.Nonetheless, a previous study developed a BZN-loaded microemulsion and reported drastically reduced Vero cell viability (10% at 80 μg/mL) after only 12 h of contact time, clearly showing more intense cytotoxicity than the formulation developed herein (Streck et al., 2016).

Trypanocidal activity
At the highest concentration (125 μg/mL), BZN and NLC-BZN induced a 100% reduction in the viability of the BZN-resistant Colombian strain of T. cruzi (Soeiro et al., 2013).No significant effect was observed in the treatment with the blank NLC (without BZN), and the viability of the epimastigotes was similar to the cell viability control (Figure 4C).Both free BZN and NLC-BZN showed a dose-dependent relationship with cell viability, although the effect of BZN encapsulation was more remarkable in concentrations above 3.90 μg/ mL (p < 0.05).These data suggest that encapsulation in nanoparticles increased BZN activity in this T. cruzi strain.The IC 50 for the free BZN against this strain (26.7 ± 0.1 μM) was comparable to the previous reports: 14.9-26.8μM (Vinuesa et al., 2017), 25.4 ± 2.7 μM (Moreno et al., 2010), 34.1 ± 0.8 μM (Zingales et al., 2015), and 4.1 ± 1.1 μg/mL (Meira et al., 2015).The IC 50 was lower for NLC-BZN (20.2 ± 0.4 μM), which is considered more effective than free BZN against this BZN-resistant strain, thus highly promising for further in vivo research.Lipid nanoparticles (NLC) loaded with BZN were screened and developed with favorable physicochemical characteristics for oral and parenteral administration.
High EE and drug loading were obtained, superior to previously reported lipid systems.The FTIR, thermal, and crystallinity studies indicated nanoparticle formation with high drug encapsulation.Compared to the free BZN, the developed BZN-loaded NLC showed lower toxicity for membranes and fibroblast cells, which was associated with increased trypanocidal efficacy against the BZNresistant Colombian strain of T. cruzi.Therefore, this novel formulation has promising potential in Chagas disease treatment and must be further studied in more complex models, such as in vivo experiments.

FIGURE 2 -
FIGURE 2 -FTIR spectra of BZN and the pure formulation components (a); lyophilized formulation (blank and loaded with BZN), and the physical mixture (b).BZN main peaks were highlighted in the respective spectrum.

FIGURE 3 -
FIGURE 3 -Diffractograms of the pure components and lyophilized formulations (blank and loaded with BZN) in the thin film (TF) and normal (Norm) experiments (a).DSC curves of the pure components and lyophilized formulations obtained in nitrogen atmosphere (b).TG curves of the pure components and lyophilized formulations obtained in nitrogen atmosphere (c).

FIGURE 4 -
FIGURE 4 -Percentage of hemoglobin release after contact of RBC with BZN, NLC-BZN, and b-NLC (a).Viability of L929 cells after 72 h of treatment with BZN and NLC-BZN (b).Cell viability of T. cruzi epimastigotes after 72 h of treatment with BZN, NLC-BZN, and b-NLC (c).
. The NLC components were Compritol 888 ATO kindly provided by Gattefossé (Lyon, France) and Super Refined Tween 80 and Crodamol GTCC kindly provided by Croda Inc.

TABLE I -
Composition