Synthesis, Characterization, and Activity of Hydroxymethylnitrofurazone Nanocrystals against Trypanosoma cruzi and Leishmania spp.
by
, ,
Cauê Benito Scarim
1,*
,
Aline de Souza
2,
Débora Soares Souza Marins
2,
Elda Gonçalves dos Santos
3,
Lívia de Figueiredo Diniz Castro
3,
Ivo Santana Caldas
3,
Patrícia Ferreira Espuri
3,
Marcos José Marques
3,
Elizabeth Igne Ferreira
2,
Nadia Araci Bou-Chacra
2 and
Chung Man Chin
1,4 1
Department of Drugs and Medicines, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara 14800-903, SP, Brazil
2
Department of Pharmacy, School of Pharmaceutical Sciences, São Paulo University (USP-SP), São Paulo 05508-000, SP, Brazil
3
Department of Pathology and Parasitology, Federal University of Alfenas (UNIFAL-MG), Alfenas 37130-000, MG, Brazil
4
Advanced Research Center in Medicine, School of Medicine, Union of the Colleges of the Great Lakes (UNILAGO), São José do Rio Preto 15030-070, SP, Brazil
*
Author to whom correspondence should be addressed.
Drugs Drug Candidates 2022, 1(1), 43-55; https://doi.org/10.3390/ddc1010005
Submission received: 20 October 2022
/
Revised: 1 December 2022
/
Accepted: 1 December 2022
/
Published: 13 December 2022
(This article belongs to the Collection Anti-Parasite Drug Discovery)
Abstract
:Hydroxymethylnitrofurazone (NFOH) is a prodrug of nitrofurazone devoid of mutagenic toxicity, with in vitro and in vivo activity against Trypanosoma cruzi (T. cruzi) and in vitro activity against Leishmania. In this study, we aimed to increase the solubility of NFOH to improve its efficacy against T. cruzi (Chagas disease) and Leishmania species (Leishmaniasis) highly. Two formulations of NFOH nanocrystals (NFOH-F1 and NFOH-F2) were prepared and characterized by determining their particle sizes, size distribution, morphologies, crystal properties, and anti-trypanosomatid activities. Furthermore, cytotoxicity assays were performed. The results showed that the optimized particle size of 108.2 ± 0.8 nm (NFOH-F1) and 132.4 ± 2.3 nm (NFOH-F2) increased the saturation solubility and dissolution rate of the nanocrystals. These formulations exhibited moderate anti-Leishmania effects (Leishmania amazonensis) in vitro and potent in vitro activity against T. cruzi parasites (Y strain). Moreover, both formulations could reduce parasitemia (around 89–95% during the parasitemic peak) in a short animal model trial (Y strain from T. cruzi). These results suggested that the increased water solubility of the NFOH nanocrystals improved their activity against Chagas disease in both in vitro and in vivo assays.
1. Introduction
American trypanosomiasis (Chagas disease) and leishmaniasis are considered neglected tropical diseases (NTDs) and represent a severe global health problem. These two NTDs, caused by the protozoans Trypanosoma cruzi and Leishmania spp., respectively, affect approximately 10 million people worldwide and are responsible for more than 44,000 deaths per year [1,2]. Available treatments for these NTDs still rely on decades-old drugs. Most of these drugs are toxic, show decreasing efficacy, and exhibit several side effects. They also involve a complicated route of administration and require patient compliance [3,4,5,6,7,8,9,10,11]. Therefore, discovering and developing novel, effective, safe, and affordable anti-trypanosomatid agents are a high priority [12,13].
Hydroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug (Figure 1), which has been considered a crucial and promising anti-T. cruzi candidate in vitro [14,15]. Its activity was evaluated and confirmed in murine models with acute and chronic stages of Chagas disease [15,16,17]. Moreover, the Ames test confirmed the absence of mutagenic toxicity in NFOH [14]. Furthermore, no hepatotoxicity was presented by NFOH when administered in Balb/c mice for a long period (60 days) in high concentrations (150 mg kg) [17,18].
Developing nanocrystal-based pharmaceutical products has increased in the last few decades. The Food and Drug Administration received more than 80 submissions of nanocrystal-containing drugs by the year 2016 that could be administered by different routes and for several diseases [19]. Nanocrystals are carrier-free drug particles prepared in dispersion media using stabilizers (surfactants or polymers), resulting in a colloidal system [20]. The stabilization (steric, electrostatic, or electrosteric) can minimize the risk of nanoparticle agglomeration. These nanoparticles can improve the dissolution velocity and increase the saturation solubility of the drugs based on Noyes–Whitney equations [21]. Additionally, nanocrystals can increase the adhesiveness of the biological membranes and the surface of the gastrointestinal tract compared with micrometer particles [20].
The preparation and physicochemical characterization of the NFOH nanocrystals (NFOH-F1 and NFOH-F2) presented in this study were proposed as an approach to obtain active targeted drug delivery against T. cruzi (Y strain) and Leishmania amazonensis (L. amazonensis), improving their in vitro activity against these trypanosomatid parasites. Moreover, due to potential anti-T. cruzi effects, the Chagas-murine model was evaluated for five day-treatment.
2. Results and Discussion
2.1. Screening for the Preparation of NFOH Nanocrystals
Formulations were screened based on the presence of monomodal distribution and the presence of precipitates, as they indicate the physical stability of the nanosuspension. Formulations F1 (particle size = 108.2 ± 0.8 nm) and F2 (particle size = 132.4 ± 2.3 nm) showed a monomodal distribution. The polydispersity indices (PI) for F1 and F2 were 0.281 ± 0.015 and 0.270 ± 0.002, respectively. Thus, the formulations F1 and F2 were accepted, and F3, F4, F5, and F6 were rejected. Poloxamer 188 (P188) and poloxamer 407 (P407) surfactants were used to prepare NFOH-F1 and NFOH-F2 nanocrystals, respectively.
2.2. Factorial Design
Eleven experiments were performed corresponding to a factorial design employing three two-level factors (23) to evaluate the effect of independent variables. Table 1 shows the mean particle size (MPS), PI, and zeta potential (ZP) results obtained for these 11 experiments using P407. MPS and PI ranged from 191.3 ± 2.1 to 326.8 ± 4.6 nm and 0.21 ± 0.01 to 0.50 ± 0.01, respectively. ZP ranged from −23.9 ± 0.7 to −30.8 ± 2.2 mV.
Table 2 shows the analysis of variance (ANOVA) performed, and parameters with p < 0.05 (α = 0.05) were considered significant. The linear model was significant (p < 0.05; α = 0.05). Among the main factors, only the stirring time was significant (p = 0.001; α = 0.05). The following interactions were significant: NFOH*P407, NFOH*Stirring time, and P407*Stirring time (p < 0.05, α = 0.05). The coefficients revealed similar values: 99.36%, 97.86%, and 98.36%, respectively, for R-square (R2), adjusted-R2 [R2 (adj)], and predicted R2 [R2 (pred)] of the adjusted model.
The assumption of normality was verified using the normal probability plot, residual versus adjustment graph, and histogram. The residual versus adjustment graph showed randomness in the distribution.
The model was described by Equation (1), which correlates the effect of each factor and their interactions with the MPS response. Two experiments were performed using different concentrations to verify the adequacy of the model equation (A) NFOH (2.5% m/m) and P407 (2.5% m/m) and (B) NFOH (1.5% m/m) and P407 (1.5% w/w), as shown on Table 3.
Equation (1).
MPS = 559.8 − 82.89 NFOH − 81.93 P407 − 2.919 Agitation Time + 18.78 NFOH × P407 + 0.5118 NFOH × Agitation Time + 0.6 P407 × Agitation Time
For P188, the same analyses were performed; however, the linear model did not adequately fit the data. Therefore, performing another statistical analysis to design an appropriate mathematical model for evaluating the experimental data was necessary in which 6 axial points and 3 central points were added in the factorial design. Thus, the factors considered significant in the new analysis were NFOH concentration, stirring time, and P188 concentration.
The results of the response surface study are shown in Table 4. The MPS ranged from 184.8 ± 0.5 to 325.9 ± 2.2 nm, PI from 0.21 ± 0.01 to 0.57 ± 0.01, and ZP ranged from −31.7 ± 1.1 to −40.3 ± 0.6 mV.
Table 5 shows the ANOVA results for the MPS response. The quadratic model presented p < 0.05 (α = 0.05). The coefficients revealed similar values: 98.00%, 97.29%, and 95.30%, respectively, for R2, R2 (adj), and R2 (pred) of the adjusted model. Furthermore, the lack of adjustment was not significant (0.051; α = 0.05); therefore, the obtained model was considered statistically significant. The normal probability plot for residuals was approximately linear, revealing a normally distributed behavior. No heteroscedasticity phenomenon was observed. The graph of the individual observations showed the random behavior of the residuals (data not shown).
The model was described by Equation (2), which correlated the effect of each factor and their interactions with the MPS response. The coefficient for the term agitation time (h) contributed more efficiently to MPS reduction compared with NFOH concentration (% w/w), which was proved by the interaction of two factors (P188 [% w/w]*P188 [% w/w]) with negative coefficients.
Two experiments were performed using the following different concentrations to verify the adequacy of Equation 2 of the model: NFOH (2.5% w/w) and P188 (2.5% w/w) for C and NFOH (1.5% m/m) and P188 (1.5% m/m) for D, both with 120 h of stirring. Under these conditions, the MPS values observed in Table 5 were within the predicted value range, calculated with α = 0.05. Thus, the result corroborated the validity of the mathematical model for MPS. The formulas selected for continuing the study are described in Table 6. They were selected because of their stability, and the MPS was less than 200 nm.
Equation (2).
MPS = 188.2 − 8.35 NFOH + 224.6 P188 − 1.3728 Agitation Time − 55.78 P188 × P188
Comparison between nanoparticle tracking analysis (NTA) and photon correlation spectroscopy (PCS) for particle size determination of NFOH nanocrystals. NTA is a technique for the visualization and direct real-time analysis of nanoparticles in liquids. Table 7 and Figure 2 show the particle sizes of the nanocrystals of NFOH.
2.3. X-ray Diffraction (XRD)
NFOH crystallinity was previously determined by Doriguetto et al. (2005) [22]. XRD of NFOH and its nanosuspensions (Figure 3) showed characteristic peaks at 9.5°, 21.2° and 28.1° 2θ. Lyophilized NFOH nanocrystals characteristic peaks remained unchanged in the diffractograms with a decrease in peak height. The peak height is affected by crystal size and crystallinity [23]. These results showed that the adapted method of wet bead milling method was appropriated to prepare NFOH nanosuspensions.
3. Biological Activity
3.1. Cytotoxicity on Macrophages (50.0% Cytotoxicity Concentrations [CC50])
Both formulations presented low cytotoxicity to mouse peritoneal macrophage cells at tested concentrations (Table 8 and Table 9). The CC50 values for peritoneal macrophage cells were 1194.7 and 885.5 µM for NFOH-F1 and NFOH-F2, respectively (Table 8 and Table 9). The selectivity index analysis (SI = CC50/50.0% inhibitory growth concentration [IC50]) (Table 8 and Table 9), which measured the selective toxicity extent of a compound to the parasites compared with a toxicity extent to the peritoneal macrophage cells of mice, showed that all evaluated compounds were more selective to the parasites than to the peritoneal macrophage cells.
3.2. Anti-T. cruzi Activity (In Vitro)
The NFOH-F1 and NFOH-F2 formulations exhibited a high inhibition capacity, with IC50 values of 11.02 µM for NFOH-F1 (SI = 108) and 18.83 µM for NFOH-F2 (SI = 47) in T. cruzi epimastigote forms (Y strain, IC50 BZN = 8.01 µM) (Table 8). Moreover, NFOH-F1 and NFOH-F2 showed interesting values (IC50 = 8.9 and 10.5 µM; SI = 134 and 84, respectively) against the blood trypomastigote forms of T. cruzi (Y strain) compared to frontline drug benznidazole (BZN) (IC50 BZN = 4.21 µM) (Table 8).
Both formulations (NFOH-F1 and NFOH-F2) presented potent values of IC50 (2.29 and 4.85 µM) and SI (521 and 182) against amastigote forms (Y strain), which showed promising results when compared with that of the standard drug BZN (IC50 = 4.8 µM) (Table 8). These are promising data considering the relevance of trypomastigote and amastigote human forms for treating Chagas disease.
3.3. Anti-Leishmanial Activity (In Vitro)
Both the formulations (NFOH-F1 and NFOH-F2) were evaluated against Leishmania ssp (Table 9). The tested formulations (NFOH-F1 and NFOH-F2) exhibited moderate activity against the promastigote (IC50 = 101.97 and 98.65 µM, SI = 11.72 and 8.97) and amastigote (IC50 = 90.35 and 143.64 µM, SI = 7.71 and 16.01) forms of L. amazonensis compared with amphotericin B (Table 9). Therefore, these formulations showed low cytotoxicity (1194.7 and 885.5 µM for NFOH-F1 and NFOH-F2, respectively) compared with amphotericin B (27.05 µM, Table 9). These data are important considering the relevance of current leishmania therapy.
3.4. Anti-T. cruzi Activity in Mice
Both formulations (NFOH-F1 and NFOH-F2) were administered to mice orally by gavage in a 5% Cremophor (Sigma) vehicle. Parasitemia was reduced in 100% of the animals treated with NFOH-F1 and NFOH-F2 or BZN. The area under the curve and parasitemia peak were decrease in the group treated with NFOH-F1, and NFOH-F2 could be clearly observed during treatment (Figure 4). Mice receiving NFOH-F1 and NFOH-F2 showed a parasitemia peak with an order less than that of BZN (Figure 4). The animals showed normal habits, and no death was observed during the protocol.
We observed the improved anti-trypanosomatid activity after NFOH nanocrystal administration. Extensive studies have shown that nanosystems have the potential to be used for treating leishmaniasis [24] and Chagas disease [25]. The solubility and permeation of NFOH-F1 and NFOH-F2 were improved because of the increased superficial area, leading to increase bioavailability. A recent study using NFOH in another nanostructured polymeric system showed that it was 95-fold more active and exhibited SI values of about 50-fold compared with free NFOH when assayed against L. amazonensis amastigotes [26].
Both nanocrystal formulations (NFOH-F1 and NFOH-F2) exhibited anti-T. cruzi and anti-L. amazonensis activities in vitro. The effect against T. cruzi was potent and had similar IC50 values on all forms of the parasite, especially the amastigote forms. For these latter, the IC50 values were promising for NFOH-F1 and NFOH-F2 compared to BZN (Table 8), which were consequently tested in murine models. The in vivo results showed the ability of the nanocrystal formulations to eliminate blood parasitemia completely in the infected mice (Y strains-T. cruzi) (Figure 4). Thus, future studies should emphasize evaluating the capacity of these formulations to act on both stages of Chagas disease with low toxicity to consider NFOH as a safe drug.
4. Material and Methods
4.1. Chemistry
4.1.1. Preliminary Assay for Preparation of NFOH Nanocrystals
The nanocrystals of NFOH were prepared by the wet bead milling method adapted by Romero et al. (2016) [27]. The formulas were prepared using a small-scale system consisting of a glass grinding vessel (10 mL capacity), 0.1 mm (7.5 g) zirconium oxide beads, and three cross bars. Different steric stabilizers were tested for preselecting those that allowed obtaining a preparation with MPS < 300 nm, PI < 0.3, and monomodal distribution immediately after preparation. Six formulas were prepared with 3.0% (w/w) of NFOH and 1.0% (w/w) of different surfactants (Polysorbate® 80 [Sigma-aldrich, San Luis, Missouri–EUA], Polysorbate® 20 [Sigma-aldrich, San Luis, Missouri–EUA], P188, P407, Kolliphor® HS 15 [Basf, Ludwigshafen–Germany], and Povacoat® [Daido Chemical Corporation, Osaka–Japan]) for 120 h at 800 rpm. The pH values of all formulas were adjusted to 7.5 using NaOH 0.1 M solution.
4.1.2. Factorial Design
After the preliminary tests, a factorial design was used to determine the important variables of the wet bead milling method and to obtain an appropriate mathematical model to describe the phenomenon. Two stabilizers, P188 and P407, were selected in the pre-selection stage. The independent variables selected were agitation time (h), NFOH concentration (% w/w), and surfactant concentration (% w/w). The responses or dependent variables were MPS, PI, and ZP. The amount of zirconium oxide beads (7.5 g) and the stirring speed (800 rpm) were constant for all formulations. The statistical analysis of the results obtained in the factorial design was performed using Minitab® 17.
4.1.3. MPS and PI Analysis
MPS and IP were determined using Zetasizer ZS90 (Malvern Instruments, Malvern, UK) by PCS at 25 °C and 90 °C. The samples were diluted in water to a suitable concentration. The particle size was determined by NTA. The samples were diluted in a saturated NFOH solution until the concentration of 4 nL/mL was achieved. Approximately 500 µL of this solution was injected into the NanoSight NS300 (Malvern Panalytical, UK) sample holder. Experiments were performed in triplicate with a duration of 60 s at a temperature of 25 °C using NTA software v3.1.
4.1.4. ZP
ZP was determined using Zetasizer ZS90 (Malvern Instruments, Malvern, UK) by the electrophoretic mobility method. The applied field force was 20 V/cm. ZP measurements were calculated in purified water with a conductivity adjusted to 50 µS.cm−1 by adding 0.9% NaCl (w/v).
4.1.5. Lyophilization
Lyophilization was performed as follows: freezing the formulations at −45.0 °C, annealing with heating at −15 °C, and maintaining at that temperature for 1 h; primary drying at −30 °C shelf temperature and 75 mTorr pressure for 48 h and secondary drying at +25 °C shelf temperature under the same pressure for 4 h. The lyophilized formulations were used for biological activity assays.
4.1.6. XRD
XRD was performed to detect the crystallinity of pure NFOH and lyophilized NFOH nanosuspension. The powder was placed in a sample holder and scanned between 10° and 80° (2θ), exposed to CuKa radiation 40 kV and step time of 0.01.
4.2. Biology
4.2.1. Cytotoxicity on Macrophages (CC50)
A suspension of 8 × 105 murine peritoneal macrophages in RPMI 1640 medium and bovine fetal serum (BFS) at 10.0% (inactivated by heat treatment), besides 1.0% penicillin (10,000 UI/mL)/streptomycin (10 mg/mL), which were placed to each well in 96-well plates. For cell adhesion, the macrophages plated were incubated in 5.0% CO2 air mixture at 37 °C. Non-adherent cells were removed, after 24 h, by washing with RPMI 1640 medium. Then, compounds (NFOH-F1 and NFOH-F2) and reference drugs were added to the wells containing the cells in different concentrations, ranging from 3.91 to 500.00 µg/mL in dimethyl sulfoxide (DMSO) with a final concentration of 0.6% (v/v), and the plates were incubated for another 48 h. Non-adherent cells were removed by washing with the RPMI 1640 medium. Then, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was solubilized in phosphate-buffered saline (5.0 µg/mL), and 10 µL of it was added to the RPMI 1640 medium to make up a volume of 200.0 µL per well, followed by an incubation for 4 h [28]. Subsequently, the medium was removed, and 100.0 µL of DMSO was added to each well and homogenized for 15 min. Afterward, the absorbance of each well was calculated at 570 nm according to Equation (3) (OD represents optical density).
Equation (3).
Inhibition = (ODcontrol − ODcompounds/ODcontrol) × 100
Each experiment was performed in triplicate on three different days, and the cell culture control (medium + cells + DMSO 0.6% v/v) was used to calculate the percentage of viable cells and the determination of CC50 values. The ratio of CC50 and IC50 values for amastigote forms were used to determine the selective index (SI).
4.2.2. In Vitro anti-T. cruzi Activity (IC50)
The stock solutions of NFOH-F1, NFOH-F2, and BZN were solubilized in DMSO at 10 mg/mL. The epimastigotes (1.5 × 106/mL per well) were incubated (BOD Incubators model) at 28 °C for 72 h. The analysis of those compounds were done in triplicate of seven doses, 200 µg/mL and a further six 2-fold serial dilutions. No effect against the epimastigotes was observed by the final DMSO concentration. Resazurin (1 mM) was added to each well, after incubation period. Absorbance at 570 nm and 600 nm were measured after 12 h of incubation with resazurin. The formula used to calculate the percentage of the epimastigotes proliferation inhibition was the following: % inhibition = 100 − [A570 − (A600 × R0) Treated/A570 − (A600 × R0)Control+] × 100, where A570 = absorbance at 570 nm, A600 = absorbance at 600 nm, Control+ = positive control: well containing culture medium, resazurin and epimastigotes without treatment, Control- = negative control: well containing only the culture medium and resazurin, without epimastigotes and treatment; R0 = absorbance of the negative control [R0 = (A570/A600)Control−] [29]. For the antitrypomastigote assay, the parasites (1 × 106) were incubated for 24 h, with the same concentrations of the evaluated compounds. The rates of parasite death were quantified by light microscopy. The IC50 values of the compounds were calculated for both antiepimastigote and antitrypomastigote assays at the CalcuSyn software (Biosoft, Cambridge, UK).
For analyzing the effects of NFOH-F1, NFOH-F2, or BZN against intracellular parasites in a 72 h assay, cells from neonatal rat cardiomyoblasts (1 × 104 cells) of the H9c2 lineage (American Type Culture Collection, ATCC: CRL 1446) were incubated for 24 h in DMEM medium supplemented with 0.2% gentamicin (200 µg/mL), 1% glutamine 2 nM, and 10% FBS). After that, the cells were infected with trypomastigotes at a ratio of 20 parasites to 1 H9c2 cell and the plate were incubated for 24 h. Then, the treatment with the compounds NFOH-F1, NFOH-F2, or BZN at the following concentrations 120 µM, 60 µM, 30 µM, 15 µM, 7.5 µM, 3.75 µM, and 1.87 µM were done for three days. All tissue culture plate incubations were done at 37 °C and in a 5% CO2-air mixture. After that, the cultures were fixed with methanol, stained with Giemsa, and the percentages of infected cells were estimated through a microscopy analysis of the treated and untreated cultures. The percentage of growth inhibition was calculated by the following formula: number of infected cells in the treated cultures / mean value of infected cell in the infected control cultures × 100. [30]. The IC50 values were calculated using the CalcuSyn software.
4.2.3. In Vitro Anti-Leishmania Activity (IC50)
The promastigotes of L. amazonensis (strain MHOM/BR/71973/M2269) were cultivated on 24-well plates in LIT medium supplemented with BFS at 10.0% (inactivated by heat treatment) and 1.0% penicillin (10,000 UI/mL)/streptomycin (10.0 mg/mL) (Sigma, USA). In the logarithmic phase of the proliferation curve, cells were harvested and suspended in fresh medium, counted using Neubauer chambers, and adjusted to a concentration of 1 × 106 cells/mL using 24-wells plates. The compounds NFOH-F1 and NFOH-F2, in concentrations ranging from 0.10–40.00 µg/mL, were solubilized in DMSO (0.6% v/v in all wells), added to the promastigote cultures (1 × 106 cells/mL), and incubated at 25 °C. The remaining parasites were counted in a Neubauer chamber and compared with controls DMSO (0.6% v/v) to calculate IC50 [31], after 72 h of incubation. Amphotericin B (Sigma) was used as a reference drug and all tests were performed in triplicate at three different times.
Murine peritoneal macrophages were cultivated in RPMI 1640 medium (Sigma, USA) supplemented with BFS at 10.0% (inactivated by heat treatment) at 37 °C in 5.0% CO2 incubator (project number 59/2017) for the assays with amastigote forms. In a 24-well plate chamber plus 13-mm glass slides (Nunc, Thermo Fischer Scientific Massachusetts-USA), the cells with a density of 8 × 105 cells per well were cultured. The macrophages were infected with late log-phase promastigotes at a ratio of 10:1 (parasite/macrophage), followed of incubation at 37 °C in 5.0% CO2 incubator for 24 h (Espuri et al., 2019). Non-phagocytosed promastigote forms were removed by washing, and NFOH-F1 and NFOH-F2, in the concentrations 0.10–40.00 µg/mL, were administered after solubilization in DMSO at a concentration of 0.6% v/v. Absolute methanol was used, after 72 h, to fix the cells in the chamber slide, which were stained with 10.0% Giemsa, followed by examination in optical light microscope plus oil immersion. At least 200 macrophages were counted for the calculation of the percentage of infected cells per well. The ratio of inhibition (IC50 value) was calculated regarding the control with DMSO. Amphotericin B (Sigma) was used as a reference drug and all tests were performed in triplicate at three different times.
4.2.4. Anti-T. cruzi Murine Assay
Female Swiss mice (N = 20; n = 5) at about five weeks of age (20–25 g) were purchased from the Universidade Federal de Alfenas. The experiment was done in an ambient with control of temperature and water and food ad libitum. Blood trypomastigotes (5 × 103) of the T. cruzi Y strain were intraperitoneally inoculated. T. cruzi Y strain is known to have induce high level of parasitemia, 100% mortality when non-treated, and to be partially resistant to BZN. Tail blood was examined for the presence of parasites at four days post-infection (dpi). Only when trypomastigotes were detected in microscopy analysis, was the treatment started (5 dpi) [30]. The treatment drug was administered orally at 100 mg kg−1. NFOH-F1, NFOH-F2, and BZN were diluted using distilled water with 5% de Cremophor (Sigma). The animals were treated by 200 µL of the compound suspensions through gavage at 5–9 dpi. Parasitemia and mortality were checked every day until 15 dpi. The protocol was approved by the Research Ethics Commission of the UNIFAL-MG (project number 59/2017) and was performed per the Guide for the Care and Use of Laboratory Animals.
4.3. Statistical Analysis
The 5% significance level (p < 0.05) was determined by Tukey multiple comparisons and ANOVA. GraphPad Prism (v. 8.1) was used for ANOVA.
5. Conclusions
The NFOH nanocrystals described in the present study (NFOH-F1 and NFOH-F2) exhibited similar activities compared with free NFOH [14,15,16,17]. Likewise, considering the fact that the NFOH nanocrystals can be tested against other strains of T. cruzi or Leishmania in animal assays, the present results are important to future studies on NFOH (free, nanocrystals, or polymers). Therefore, future studies with these potent novel anti-parasitic formulations should involve assays in both stages of Chagas disease and toxicity assays in mice to assess their cytotoxicity.
In conclusion, NFOH-F1 and NFOH-F2 exhibit similar effects as free NFOH and BZN, indicating their potent effects on the short acute stage of experimental Chagas disease in a murine model. Thus, both formulations (NFOH-F1 and NFOH-F2) can be promising drug candidates for future analysis against trypanosomatid parasites.
Author Contributions
C.B.S., A.d.S., D.S.S.M., E.G.d.S., L.d.F.D.C., I.S.C., P.F.E., M.J.M., E.I.F., N.A.B.-C. and C.M.C. designed the experiments and analyzed the data. C.B.S., A.d.S., D.S.S.M., E.G.d.S., L.d.F.D.C. and P.F.E. performed the experiments. C.B.S. wrote the manuscript, with input from I.S.C., M.J.M., E.I.F., N.A.B.-C. and C.M.C. All authors have read and agreed to the published version of the manuscript.
Funding
The authors would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-2016/10847-9) for assistance in the form of research fellowships. This work was financed in part by the Coordenação de Aperfeiçoamento Pessoal de Nível Superior-Brasil (CAPES), finance code: 001.
Institutional Review Board Statement
This study was carried out in strict accordance with the recommendations in the UNIFAL Guide for the Care and Use of Laboratory Animals.
Informed Consent Statement
Not applicable.
Data Availability Statement
The data supporting the findings of the research article is available within this article.
Acknowledgments
The authors would like to thank the Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP (PADC/FCF-UNESP).
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Chemical structure of hydroxymethylnitrofurazone (NFOH).
Figure 2.
Hydroxymethylnitrofurazone (NFOH) nanocrystals by nanoparticle tracking analysis. (A): F-P188 (NFOH-F1); (B): F-P407 (NFOH-F2).
Figure 2.
Hydroxymethylnitrofurazone (NFOH) nanocrystals by nanoparticle tracking analysis. (A): F-P188 (NFOH-F1); (B): F-P407 (NFOH-F2).
Figure 3.
Diffractogram: (A) NFOH-1 (F1), NFOH, poloxamer 188 (P188), and mannitol; (B) NFOH-2 (F2), NFOH, poloxamer 407 (P407), and mannitol.
Figure 3.
Diffractogram: (A) NFOH-1 (F1), NFOH, poloxamer 188 (P188), and mannitol; (B) NFOH-2 (F2), NFOH, poloxamer 407 (P407), and mannitol.
Figure 4.
Effect on parasitemia of treatment with hydroxymethylnitrofurazone (NFOH)-F1, NFOH-F2, and BZN. The means of parasitemia values are represented by the lines. Yellow area: treatment period (5 days). POS: mice infected by T. cruzi and untreated; BZN: mice infected by T. cruzi and treated by BZN; NFOH-F1: mice infected by T. cruzi and treated by NFOH-F1; NFOH-F2: mice infected by T. cruzi and treated by NFOH-F2; Peak value: parasitemic peak (parasites); AUC: area under the curve; ** significant difference (p < 0.05).
Figure 4.
Effect on parasitemia of treatment with hydroxymethylnitrofurazone (NFOH)-F1, NFOH-F2, and BZN. The means of parasitemia values are represented by the lines. Yellow area: treatment period (5 days). POS: mice infected by T. cruzi and untreated; BZN: mice infected by T. cruzi and treated by BZN; NFOH-F1: mice infected by T. cruzi and treated by NFOH-F1; NFOH-F2: mice infected by T. cruzi and treated by NFOH-F2; Peak value: parasitemic peak (parasites); AUC: area under the curve; ** significant difference (p < 0.05).
Table 1.
Matrix of the response surface test using poloxamer 407 as a surfactant for mean particle size, polydispersity index, and zeta potential of the hydroxymethylnitrofurazone nanocrystals.
Table 1.
Matrix of the response surface test using poloxamer 407 as a surfactant for mean particle size, polydispersity index, and zeta potential of the hydroxymethylnitrofurazone nanocrystals.
| Formula | Order | Central Point | Time (hours) | NFOH (%w/w) | P407 (%w/w) | MPS | PI | ZP |
|---|---|---|---|---|---|---|---|---|
| 1 | 1 | 1 | 48 | 3.0 | 1.0 | 248.2 ± 1.2 | 0.39 ± 0.01 | −26.2 ± 0.5 |
| 2 | 2 | 0 | 84 | 2.0 | 2.0 | 271.0 ± 1.8 | 0.28 ± 0.02 | −27.4 ± 0.2 |
| 3 | 3 | 1 | 120 | 3.0 | 1.0 | 191.3 ± 2.1 | 0.32 ± 0.04 | −28.1 ± 1.4 |
| 4 | 4 | 0 | 84 | 2.0 | 2.0 | 278.3 ± 2.4 | 0.24 ± 0.01 | −32.1 ± 2.3 |
| 5 | 5 | 1 | 48 | 3.0 | 3.0 | 254.1 ± 6.7 | 0.41 ± 0.06 | −25.5 ± 1.5 |
| 6 | 6 | 1 | 120 | 1.0 | 1.0 | 197.2 ± 0.5 | 0.25 ± 0.01 | −30.8 ± 2.2 |
| 7 | 7 | 1 | 120 | 1.0 | 3.0 | 214.4 ± 1.4 | 0.24 ± 0.02 | −23.9 ± 0.7 |
| 8 | 8 | 0 | 84 | 2.0 | 2.0 | 264.0 ± 1.1 | 0.28 ± 0.01 | −27.7 ± 0.2 |
| 9 | 9 | 1 | 120 | 3.0 | 3.0 | 284.6 ± 1.9 | 0.21 ± 0.01 | −28.0 ± 1.2 |
| 10 | 10 | 1 | 48 | 1.0 | 1.0 | 326.8 ± 4.3 | 0.21 ± 0.01 | −29.1 ± 0.9 |
| 11 | 11 | 1 | 48 | 1.0 | 3.0 | 258.6 ± 2.8 | 0.50 ± 0.01 | −27.2 ± 0.8 |
Table 2.
Analysis of variance (ANOVA) for mean particle size using poloxamer 407 Abbreviations: DF = degree of freedom, SS = sequential sums of squares, MS = sequential mean squares, F-Value = value of the F distribution, and p-value = lack-of-fit adjustment.
Table 2.
Analysis of variance (ANOVA) for mean particle size using poloxamer 407 Abbreviations: DF = degree of freedom, SS = sequential sums of squares, MS = sequential mean squares, F-Value = value of the F distribution, and p-value = lack-of-fit adjustment.
| DF | SS (adj) | MS (adj) | F Value | p Value | ||
|---|---|---|---|---|---|---|
| Model | 7 | 15,890.7 | 2270.10 | 66.27 | 0.003 | |
| Linear | 3 | 5344.6 | 1781.53 | 52.01 | 0.004 | |
| NFOH %w/w | 1 | 44.2 | 44.18 | 1.29 | 0.330 | |
| P407 %w/w | 1 | 290.4 | 290.41 | 8.48 | 0.062 | |
| Stirring Time (h) | 1 | 5010.0 | 5010.00 | 146.26 | 0.001 | |
| Interaction | 3 | 92,268.3 | 3089.44 | 90.19 | 0.002 | |
| NFOH %w/w × P407 %w/w | 1 | 2820.0 | 2820.00 | 82.33 | 0.003 | |
| NFOH %w/w × Stirring Time (h) | 1 | 2715.8 | 2715.85 | 79.29 | 0.003 | |
| P407 %w/w × Stirring Time (h) | 1 | 3732.5 | 3732.48 | 108.97 | 0.002 | |
| Curve | 1 | 1277.8 | 1277.76 | 37.30 | 0.009 | |
| Error | 3 | 102.8 | 4.25 | |||
| Lack of adjustment | 1 | 0.5 | 0.50 | 0.01 | 0.930 | |
| Pure Error | 2 | 102.3 | 51.13 | |||
| Total | 10 | 15,993.4 | ||||
| SD = 5.85263 | R2 = 99.36% | R2 (adj) = 97.86% | R2 (pred) = 98.36% | |||
Table 3.
Observed and predicted mean particle size and polydispersity index results from the hydroxymethylnitrofurazone nanocrystals.
Table 3.
Observed and predicted mean particle size and polydispersity index results from the hydroxymethylnitrofurazone nanocrystals.
| Formula | Predicted MPS (nm) | Observed MPS (nm) | PI |
|---|---|---|---|
| A | 248.39 | 245.2 ± 1.4 | 0.29 ± 0.01 |
| B | 204.67 | 199.4 ± 2.3 | 0.26 ± 0.03 |
| C | 213.6 ± 2.0 | 215.46 | 0.34 ± 0.01 |
| D | 219.1± 2.3 | 222.33 | 0.28 ± 0.03 |
Table 4.
Matrix of the response surface test using poloxamer 188 as a surfactant for mean particle size, polydispersity index, and zeta potential of the hydroxymethylnitrofurazone nanocrystals.
Table 4.
Matrix of the response surface test using poloxamer 188 as a surfactant for mean particle size, polydispersity index, and zeta potential of the hydroxymethylnitrofurazone nanocrystals.
| Formula | Order | Central | Time (hours) | NFOH (%w/w) | P188 (%w/w) | MPS (nm) | PI | PZ (mV) |
|---|---|---|---|---|---|---|---|---|
| 1 | 1 | 1 | 48 | 3.0 | 1.0 | 285.9 ± 3.3 | 0.43 ± 0.02 | −33.2 ± 0.5 |
| 2 | 2 | 0 | 84 | 2.0 | 2.0 | 304.0 ± 2.1 | 0.29 ± 0.03 | −33.4 ± 0.7 |
| 3 | 3 | 1 | 120 | 3.0 | 1.0 | 184.8 ± 0.5 | 0.34 ± 0.04 | −33.3 ± 1.1 |
| 4 | 4 | 0 | 84 | 2.0 | 2.0 | 298.3 ± 2.1 | 0.21 ± 0.01 | −40.3 ± 0.6 |
| 5 | 5 | 1 | 48 | 3.0 | 3.0 | 274.8 ± 3.1 | 0.57 ± 0.01 | −34.6 ± 1.1 |
| 6 | 6 | 1 | 120 | 1.0 | 1.0 | 193.5 ± 3.2 | 0.23 ± 0.01 | −34.5 ± 1.1 |
| 7 | 7 | 1 | 120 | 1.0 | 3.0 | 203.0 ± 0.1 | 0.26 ± 0.01 | −34.8 ± 1.5 |
| 8 | 8 | 0 | 84 | 2.0 | 2.0 | 310.1 ± 3.3 | 0.24 ± 0.02 | −36.6 ± 0.7 |
| 9 | 9 | 1 | 120 | 3.0 | 3.0 | 194.3 ± 2.1 | 0.31 ± 0.03 | −31.7 ± 1.1 |
| 10 | 10 | 1 | 48 | 1.0 | 1.0 | 296.5 ± 2.5 | 0.22 ± 0.01 | −38.7 ± 0.6 |
| 11 | 11 | 1 | 48 | 1.0 | 3.0 | 311.8 ± 2.2 | 0.39 ± 0.02 | −38.2 ± 1.2 |
| 12 | 12 | −1 | 120 | 2.0 | 2.0 | 198.5 ± 1.4 | 0.24 ± 0.04 | −34.5 ± 1.4 |
| 13 | 13 | 0 | 84 | 2.0 | 2.0 | 298.3 ± 2.1 | 0.37 ± 0.07 | −34.5 ± 0.6 |
| 14 | 14 | −1 | 120 | 2.0 | 1.0 | 171.0 ± 0.7 | 0.22 ± 0.01 | −36.3 ± 0.7 |
| 15 | 15 | −1 | 84 | 3.0 | 1.0 | 194.4 ± 1.6 | 0.25 ± 0.01 | −36.1 ± 0.6 |
| 16 | 16 | 0 | 84 | 2.0 | 2.0 | 299.3 ± 1.9 | 0.28 ± 0.02 | −39.2 ± 0.9 |
| 17 | 17 | 1 | 48 | 2.0 | 1.0 | 256.1 ± 1.4 | 0.22 ± 0.03 | −32.5 ± 0.2 |
| 18 | 18 | −1 | 84 | 3.0 | 2.0 | 261.9 ± 2.1 | 0.29 ± 0.01 | −31.7 ± 1.3 |
| 19 | 19 | 0 | 84 | 2.0 | 2.0 | 301.2 ± 2.7 | 0.32 ± 0.01 | −38.1 ± 1.2 |
| 20 | 20 | −1 | 84 | 1.0 | 1.0 | 217.9 ± 3.3 | 0.21 ± 0.02 | −31.5 ± 1.8 |
Table 5.
Analysis of variance for mean particle size using poloxamer 188 Abbreviations: DF = degrees of freedom, SS = sequential sums of squares, MS = sequential mean squares, F-Value = value on the F distribution, and * p-value = lack-of-fit adjustment.
Table 5.
Analysis of variance for mean particle size using poloxamer 188 Abbreviations: DF = degrees of freedom, SS = sequential sums of squares, MS = sequential mean squares, F-Value = value on the F distribution, and * p-value = lack-of-fit adjustment.
| DF | SS (adj) | MS (adj) | F-Value | p-Value | ||
|---|---|---|---|---|---|---|
| Model | 5 | 48,894.5 | 9778.9 | 137.46 | 0.001 | |
| Blocks | 1 | 4318.6 | 4318.6 | 60.70 | 0.001 | |
| Linear | 3 | 26,702.8 | 8900.9 | 125.12 | 0.001 | |
| NFOH % w/w | 1 | 746.1 | 746.1 | 10.49 | 0.006 | |
| P188 % w/w | 1 | 19.1 | 19.1 | 0.27 | 0.612 | |
| Stirring time (h) | 1 | 26,149.6 | 26,149.6 | 367.57 | 0.001 | |
| Quadradic | 1 | 14,030.0 | 14,030.0 | 197.21 | 0.001 | |
| P188 % w/w*P188 % | 1 | 14,030.0 | 14,030.0 | 197.21 | 0.001 | |
| Error | 14 | 996.0 | 71.1 | |||
| Lack of adjustment | 9 | 891.2 | 99.0 | 4.72 | 0.051 | |
| Pure Error | 5 | 104.8 | 21.0 | * | * | |
| Total | 19 | 49,890.5 | ||||
| SD = 8.43456 | R2= 98.00% | R2 (adj) = 97.29% | R2 (pred) = 95.30% | |||
Table 6.
Selected formulas for characterizing in vitro and in vivo studies.
| Formula | NFOH (%w/w) | Poloxamer 188 (%w/w) | Poloxamer 407 (%w/w) | Stirring Time (h) |
|---|---|---|---|---|
| NFOH-1 | 2.0 | 1.0 | - | 120 |
| NFOH-2 | 3.0 | - | 1.0 | 120 |
Table 7.
Particle size evaluation of the hydroxymethylnitrofurazone nanocrystals Abbreviations: NTA = nanoparticle tracking analysis, PCS = photon correlation spectroscopy, and MPS = mean particle size.
Table 7.
Particle size evaluation of the hydroxymethylnitrofurazone nanocrystals Abbreviations: NTA = nanoparticle tracking analysis, PCS = photon correlation spectroscopy, and MPS = mean particle size.
| NTA | MPS (nm) | ||
|---|---|---|---|
| NFOH-F1 | NFOH-F2 | ||
| d (0,1) | 91.3 ± 3.9 | 89.2 ± 0.1 | |
| d (0,5) | 136.7 ± 2.7 | 132.6 ± 6.4 | |
| d (0,9) | 188.2 ± 6.5 | 188.7 ± 4.5 | |
| Mean | 141.9 ± 0.6 | 138.2 ± 1.7 | |
| PCS | MPS | 193.5 ± 3.2 | 197.2 ± 0.5 |
Table 8.
Anti-Trypanosoma cruzi activity (IC50, µM) against epimastigote, trypomastigote, and amastigote forms (Y strain), cytotoxicity on peritoneal macrophage cells (CC50, µM), and selectivity index ([CC50/IC50]) of hydroxymethylnitrofurazone (NFOH) formulations (NFOH-F1 and NFOH-F2) and benznidazole.
Table 8.
Anti-Trypanosoma cruzi activity (IC50, µM) against epimastigote, trypomastigote, and amastigote forms (Y strain), cytotoxicity on peritoneal macrophage cells (CC50, µM), and selectivity index ([CC50/IC50]) of hydroxymethylnitrofurazone (NFOH) formulations (NFOH-F1 and NFOH-F2) and benznidazole.
| T. cruzi Y Strain | Cytotoxicity | Epimastigotes | Trypomastigotes | Amastigotes | |
|---|---|---|---|---|---|
| SI | SI | SI | |||
| CC50 | IC50 | IC50 | IC50 | ||
| Compounds | |||||
| NFOH-F1 | 1194.7 | 11.02 +/− 2.4 108 | 8.9 +/− 0.3 | 134 2.29 +/− 0.95 | 521 |
| NFOH-F2 | 885.5 | 18.83 +/− 4.06 47 | 10.5 +/− 0.56 | 84 4.85 +/− 3.91 | 182 |
| Benznidazole | >300.0 | 8.01 +/− 1.31 >37 | 4.21 +/− 0.23 | >71 4.8 +/− 1.7 | >62 |
Table 9.
Anti-leishmanial activity (IC50, µM) against promastigote and amastigote forms (Leishmania amazonensis), cytotoxicity to peritoneal macrophage cells (CC50, µM), and selectivity index (CC50/IC50) for hydroxymethylnitrofurazone formulations (NFOH-F1 and NFOH-F2) and amphotericin B.
Table 9.
Anti-leishmanial activity (IC50, µM) against promastigote and amastigote forms (Leishmania amazonensis), cytotoxicity to peritoneal macrophage cells (CC50, µM), and selectivity index (CC50/IC50) for hydroxymethylnitrofurazone formulations (NFOH-F1 and NFOH-F2) and amphotericin B.
| Leishmania | |||||
|---|---|---|---|---|---|
| Cytotoxicity | |||||
| amazonensis/ | Promastigotes | SI | Amastigotes | SI | |
| CC50 | |||||
| Compounds | |||||
| NFOH-F1 | 1194.7 | 101.97 +/− 2.41 | 11.72 | 90.35 +/− 6.58 | 7.71 |
| NFOH-F2 | 885.5 | 98.65 +/− 7.25 | 8.97 | 143.64 +/− 1.09 | 16.01 |
| Amphotericin B | 27.05 | 5.10 +/− 0.002 | 5.3 | 1.30 +/− 0.001 | 20.8 |
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Scarim, C.B.; de Souza, A.; Marins, D.S.S.; Santos, E.G.d.; de Figueiredo Diniz Castro, L.; Caldas, I.S.; Espuri, P.F.; Marques, M.J.; Ferreira, E.I.; Bou-Chacra, N.A.; et al. Synthesis, Characterization, and Activity of Hydroxymethylnitrofurazone Nanocrystals against Trypanosoma cruzi and Leishmania spp. Drugs Drug Candidates 2022, 1, 43-55. https://doi.org/10.3390/ddc1010005
AMA Style
Scarim CB, de Souza A, Marins DSS, Santos EGd, de Figueiredo Diniz Castro L, Caldas IS, Espuri PF, Marques MJ, Ferreira EI, Bou-Chacra NA, et al. Synthesis, Characterization, and Activity of Hydroxymethylnitrofurazone Nanocrystals against Trypanosoma cruzi and Leishmania spp. Drugs and Drug Candidates. 2022; 1(1):43-55. https://doi.org/10.3390/ddc1010005
Chicago/Turabian StyleScarim, Cauê Benito, Aline de Souza, Débora Soares Souza Marins, Elda Gonçalves dos Santos, Lívia de Figueiredo Diniz Castro, Ivo Santana Caldas, Patrícia Ferreira Espuri, Marcos José Marques, Elizabeth Igne Ferreira, Nadia Araci Bou-Chacra, and et al. 2022. "Synthesis, Characterization, and Activity of Hydroxymethylnitrofurazone Nanocrystals against Trypanosoma cruzi and Leishmania spp." Drugs and Drug Candidates 1, no. 1: 43-55. https://doi.org/10.3390/ddc1010005
