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Nerolidol nanospheres increases its trypanocidal efficacy against Trypanosoma evansi: New approach against diminazene aceturate resistance and toxicity
Experimental Parasitology 166 (2016) 144e149
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Nerolidol nanospheres increases its trypanocidal efficacy against Trypanosoma evansi: New approach against diminazene aceturate resistance and toxicity Matheus D. Baldissera a, *, Thirssa H. Grando a, Carine F. Souza a, Luciana F. Cossetin a, tia Nascimento b, Ana P.T. da Silva c, Daiane F. Dalla Lana d, Michele R. Sagrillo b, Ka Aleksandro S. Da Silva e, Lenita M. Stefani e, Silvia G. Monteiro a, ** a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil rio Franciscano, Santa Maria, RS, Brazil Laboratory of Cell Culture, Centro Universita c rio Franciscano, Santa Maria, RS, Brazil Laboratory of Nanotechnology, Centro Universita d Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grando do Sul (UFRGS), Porto Alegre, RS, Brazil e , SC, Brazil Department of Animal Science, Universidade do Estado de Santa Catarina, Chapeco b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Nerolidol and nanospheres present trypanocidal action in vitro against Trypanosoma evansi. Nerolidol has trypanocidal action, but without curative effectiveness in vivo. Nanoencapsulation increase curative effectiveness in vivo. Nanoencapsulation may be an interesting approach to improve the efficacy of therapeutic drugs.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2016 Received in revised form 6 April 2016 Accepted 19 April 2016 Available online 21 April 2016
The aims of this study were to develop nerolidol-loaded nanospheres, and to evaluate their efficacy in vitro and in vivo against Trypanosoma evansi, as well as to determine their physicochemical properties, morphology, and any possible side effect in vitro against peripheral blood mononuclear cell (PBMC). The nanospheres showed an adequate particle size (149.5 nm), narrow particle distribution (0.117), negative zeta potential (12.8 mV), and pH of 6.84, such as observed by transmission electron microscopy. In vitro, a trypanocidal effect of nerolidol and nanospheres containing nerolidol was observed at 0.5, 1.0, and 2.0%, i.e., both treatments showed a faster trypanocidal effect compared to chemotherapy (diminazene aceturate e D.A.). T. evansi infected mice were used to evaluate the effects of nerolidol-loaded nanospheres regarding pre-patent period, longevity, and therapeutic efficacy. Oral administration of nerolidol-loaded nanospheres at 1.0 mL/kg/day during 10 days increased mice survival (66.66%) compared to 0% and 33.33% of mice survival when treated with nerolidol in its free form and D.A., respectively. Cytotoxic study indicated that both treatments showed no side effects in vitro against PBMC, an important marker used in toxicological surveys. Therefore, nanoencapsulation increased the therapeutic efficacy of nerolidol against T. evansi, and can be used as an alternative treatment for T. evansi infection. © 2016 Elsevier Inc. All rights reserved.
Keywords: Protozoan Sesquiterpenes Transmission electron microscopy Nanomedicine
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M.D. Baldissera), [email protected] (S.G. Monteiro). http://dx.doi.org/10.1016/j.exppara.2016.04.015 0014-4894/© 2016 Elsevier Inc. All rights reserved.
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1. Introduction
2. Materials and methods
Trypanosoma evansi is a protozoan parasite that affects domestic and wild animals (Silva et al., 2002; Desquesnes et al., 2013), and rarely parasitizes humans (Joshi et al., 2005). It is considered the most widely distributed pathogenic African trypanosomes. This parasite causes a disease known as “surra”, or “Mal das Cadeiras” in horses, and it is mechanically transmitted by biting flies (Stomoxis sp., Hematopota sp., and Chrysops sp.). Diminazene aceturate (D.A.) at a single dose of 3.5 mg kg1 is capable to eliminate the parasites in bloodstream a few hours after administration. However, it has no curative efficacy since trypanosomes may pass through the bloodbrain barrier, reaching the central nervous system, as well known area of refuge to T. evansi during the residual period of drug circulation. Diminazene aceturate does not cross the blood-brain barrier in amounts sufficient to eliminate all the parasites (Masocha et al., 2007). Also, therapeutic dose of D.A. usually shows signs of toxicity, especially related to hepatotoxic and nephrotoxic effects, as reported in the literature (Baldissera et al., 2016a, 2016b). Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol), also known as peruviol, is an aliphatic sesquiterpene alcohol present in essential oils of many plants. Many medical benefits of nerolidol have been identified including antioxidant (Nogueira et al., 2013), antinociceptive (Koudou et al., 2005), anti-ulcer (Klopell et al., 2007), anti-tumor (Lapczynski et al., 2008), and anti-bacterial (Lang and Buchbauer, 2012) properties. Regarding the antiparasitic effect of nerolidol, it has shown antileishmanial (Arruda et al., 2005), antimalarial (Lopes et al., 1999), antischistosomal (Silva et al., 2014), babesicide (AbouLaila et al., 2010) and antitrypanosomal (Mohd-Shukri and Zainal-Abidin, 2011) activities. Regarding the antitrypanosomal activity, nerolidol is able to prolong life of mice infected by T. evansi, however, without curative effectiveness. Thus, we intend to use the benefits of nanotechnology to improve its curative effectiveness due capacity of nanotechnology in increase the therapeutic index, control release of the drugs and cross the blood brain barrier (Soppimath et al., 2001), as well as observed in other treatment against T. evansi described below. Also, some pharmaceuticals difficulties of nerolidol has been observed in the literature, such as solubility problems (Nogueira et al., 2013). Considering the low efficacy of drugs available for the treatment of T. evansi infection, as well as their side effects and great resistance developed by protozoan, the nanotechnology associated with vegetal species may lead to the discovery of alternative ways with appropriate efficiency to treat the disease (Alviano and Alviano, 2009). In this context, nanospheres are a specific type of nanoparticle that have been used successfully because it can protect the antiparasitic drugs from gastric inactivation, protect mucosa against drug toxicity, control drug release, improvement of bioavailability, and decrease side effects (Shukla et al., 2012). Different antiparasitic drugs have been used into nanospheres, such as bis-triazole DO870 for Trypanosoma cruzi infection (Molina et al., 2001). Encapsulated vegetal compounds have shown promising results against T. evansi such as curcumin (Gressler et al., 2015), Melaleuca alternifolia, and Achyrocline satureioides essential oils (Baldissera et al., 2014; Do Carmo et al., 2015), compared to the free form. Different antiparasitic drugs have been tested in nanospheres in order to improve their therapeutic index. To enhance the trypanocidal effect of nerolidol, we have decided to develop a nanoformulation and to test it in vitro and in vivo. Therefore, the aims of this study were to develop and characterize nanospheres containing nerolidol, and to evaluate their efficacy in vitro and in vivo against T. evansi.
2.1. Nerolidol Nerolidol (molecular weight: 222.37 g/mol) was purchased from Sigma-Aldrich® and showed 97% of purity. 2.2. Nanospheres preparation Nanospheres suspension (NS) containing nerolidol was prepared according to the interfacial deposition of preformed polymer (Fessi et al., 1989). The components of the organic phase and aqueous phase (Table 1) were placed in a beaker and kept under magnetic stirring in a water bath at 40 C for 1 h, and the active principle (AP) was added to the organic phase at the end of the heating period. With the aid of a funnel, the organic phase was poured into the aqueous phase and stirred for 10 min. This mixture was concentrated to a final volume of 100 mL in a rotary evaporator to eliminate the organic solvent, adjusting the final concentration of AP to 3 mg mL1 from pure nerolidol (100% concentration). 2.3. Nanospheres characterization The pH determination for the nanosphere suspension was performed using a potentiometer (Digimed®) previously calibrated with buffer solutions at pH 4.0 and 7.0. The determination of the diameter and polydispersity of nanoparticles in suspension were performed by dynamic light scattering using the Zetasizer®, NanoZS from Malvern equipment. The suspensions were diluted 500 times (v/v) in Milli-Q water and the results were determined by the average of three replicates. The zeta potential was obtained using the technique of electrophoretic mobility Zetasizer®, Malvern Nano-ZS instrument. The samples were pre-diluted at 500 times (v/ v) in 10 mM sodium chloride and filtered through a membrane with 0.45 mm. The results were expressed in millivolts (mV). 2.4. Scanning electron microscopy Morphological analysis was determined by transmission electron microscopy (TEM) (JEM 2010, Tokyo, Japan) at 200 KV in operation. For this, the suspension was diluted 10 in ultrapure water, placed on a specimen grid and negatively stained with uranyl acetate (2% W/V). 2.5. Trypanosoma evansi isolate This study was conducted in two consecutive experiments (in vitro and in vivo). The cryopreserved isolate of T. evansi used in these experiments was obtained from a naturally infected dog (Colpo et al., 2005). Two rats (Ra and Rb) were infected Table 1 Composition nerolidol.
of
nanospheres
suspensions
containing
Compounds mL Organic phase Eudragit RS 100® Sorbitan monostereate Nerolidol Acetone Aqueous phase Polysorbate 80 Milli-Q water
1.0 0.766 3.102 267.0 0.766 533.0
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intraperitoneally with blood contaminated by trypomastigotes, which was kept cryopreserved in liquid nitrogen. This process was performed in order to obtain a large amount of viable parasites for in vitro tests (Ra), and to infect the mice in the experimental groups (Rb). 2.6. In vitro test The culture medium for T. evansi was adapted from Baltz et al. (1985) as previously published by Baldissera et al. (2013). The protozoa were acquired from Ra three days post-infection, the rat showed high parasitemia (6.0 105 trypanosomes/mL) and it was anesthetized with isoflurane for blood collection by cardiac puncture. The blood was stored in tubes containing EDTA (ethylenediamine tetraacetic acid). For trypanosome separation, each 200 mL of blood was diluted in complete culture medium (DMEM) 1:1 (v/v), added to microcentrifuge tubes, and centrifuged at 400 g for 10 min at 25 C. The supernatant was removed and resuspended in culture medium and the number of parasites was counted in a Neubauer chamber. The culture medium containing the parasites was distributed into microtiter plates (270 mL/well), and 25 mL of each treatment was added (diluted in culture medium). For this assessment, the nerolidol and nanospheres containing nerolidol were used at concentrations of 0.5, 1.0 and 2.0%. A positive control (0.5% D.A.) was also adopted at the same volume (25 mL). Each well received 20 mL to count the number of parasites at 1, 3, 6 and 9 h after the onset of the experiment in Neubauer chambers. 2.7. Tests in vivo 2.7.1. Animal model Thirty 60-day-old female mice, heterogenic, outbred strain, weighing an average of 30 ± 0.4 g were used as the experimental model. The animals were maintained under controlled light and environment (12:12 h light-dark cycle, 23 ± 1 C, 70% relative humidity) with free access to commercial food and water. Experiments were carried out between 8 a.m. and 5 p.m. All animals were subjected to a period of 30 days for adaptation. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. These procedures were approved by the Animal Welfare Committee of Federal University of Santa Maria under protocol number 8658110915. 2.7.2. Experimental design and parasitemia estimation The mice were assigned to five groups (A - E), with six animals each. The group A consisted of uninfected and untreated animals (negative control); the group B: infected and untreated mice (positive control); the group C: animals infected and treated nerolidol 1.0 mL kg1; the group D: animals infected and treated with nanospheres containing nerolidol 1.0 mL kg1; the group E: animals infected and treated with D.A. in a single dose of 3.5 mg kg1, intramuscularly. Animals from groups B, C, D, and E were inoculated intraperitoneally with 0.06 mL of blood from Rb containing 2.0 105 trypanosomes. A daily supply of nerolidol and nanospheres containing nerolidol was maintained for five days before infection and five days after infection via oral gavage, according to the protocol used by Mohd-Shukri and Zainal-Abidin (2011). One hour post-infection, D.A. was administered according to manufactures recommendation (single dose of 3.5 mg kg1, intramuscularly). The peripheral blood from the tail of the rats was examined daily for parasitemia degree scoring system. Each slide was prepared with fresh blood collected from the tail coccygeal vein, stained by the Romanowski method, and visualized at a
magnification of 1000 according to the method described by Da Silva et al. (2006). The mice were observed for up to 60 days. 2.7.3. Treatment efficacy The number of mice that did not show clinical signs of T. evansi infection and did not die after treatment was used to determine treatment efficacy. Prepatent period, and longevity were also observed. 2.8. Cytotoxicity assay Although the natural products may be efficient, the toxicity of preparations used is usually unknown. However, toxicology tests showed that many plants currently used are highly toxic (Atsamo et al., 2011). Therefore, we decided to determine nerolidol safety by using peripheral blood mononuclear cells (PBMC) culture, once this cellular type is an indicator in vivo of damage to cells (Lexis et al., 2006). 2.8.1. PBMC cells Samples of peripheral blood were supplied by the Clinical Analysis Laboratory of the Franciscan University Center and the project was approved by this same center (CAAE: 31211214.4.0000.5306). Peripheral blood samples were collected from three apparently healthy volunteers (22e25 years old), who did not smoke, neither drank alcoholic beverages more than two times a week, nor took prescription drugs and without autoimmune or infectious diseases. The samples were collected after 12 h of overnight fasting by venipuncture using a top Vacutainer® (BD Diagnostics, Plymouth, UK) and heparin tubes. PBMC were separated by Histopaque-1077 (SigmaeAldrich Co., St Louis, USA) by density gradient centrifugation using 4 mL of blood samples. After further centrifugation for 15 min at 2.500 g, cells were transferred to culture media containing 5 mL of RPMI 1640 supplemented with 10% fetal bovine serum, and 1% of penicillin and streptomycin. The cells were cultured in a 96-well microplate at an initial density of 2 105 for 24, 48 and 72 h at 37 C in a 5% humidified CO2 atmosphere (Wilms et al., 2005). 2.8.2. Cell viability The cytotoxicity of the nerolidol and nanospheres containing nerolidol against PBMC was evaluated as described by Sagrillo et al. (2015), using the tetrazolium salt MTT 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric method based on the cleavage of the reagent by dehydrogenases in viable cells (Mosmann, 1983). In the microplates, cells were incubated at 37 C in a humid atmosphere with 5% CO2 for 24, 48, and 72 h. At the end of the incubation, 50 mL of MTT solution was added to each well of the microplate and the cells were incubated for three more hours. The supernatant was removed and 200 mL of dimethyl sulfoxide (DMSO) were added to each well. The microplate was analyzed using an ELISA reader at a wavelength of 560 nm. As a control, cells were grown in a medium lacking the constituents. The experiment was performed in triplicate. Finally, cell viability was determined, which was expressed as a percentage of the control value. 2.9. Statistical analysis The data met the assumption of parametric testing according to the Kolmogorov-Smirnov test. The bilateral two-way analysis of variance (ANOVA) followed by the Bonferroni's post hoc test were used for comparison of means. Differences between groups were rated significant at p < 0.05. All analysis were carried out in an IBM compatible computer using the Statistical Package for the Social Sciences (SPSS) software 20. Results were presented as
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mean ± standard deviation. 3. Results 3.1. Characterization of nanospheres Composition of nanospheres suspensions containing nerolidol was showed in Table 1. The nerolidol nanospheres were evaluated regarding their physical and chemical properties. The droplet size was around 149.5 nm, polydispersity of 0.117, the potential zeta was 12.8 mV and the hydrogen potential (pH) was around 6.84. TEM image show the morphology spherical of the nanospheres (Fig. 1). The mean particle size observed by TEM analysis was in agreement with those obtained by spectroscopy correlation of photons. 3.2. In vitro test The trypanocidal effect of nerolidol (Fig. 2A) and nanospheres containing nerolidol (Fig. 2B) on T. evansi was directly proportional to the concentration used. A reduction of live trypomastigotes was observed at all concentrations compared to the control group. After 6 h, there were no living trypomastigotes in 1% and 2% concentrations using nerolidol and nanospheres containing nerolidol. After 9 h of the beginning of the assay, there were also no living trypomastigotes in 0.5% concentration as well as on the D.A. treatment. On the other hand, parasites were alive in positive control samples, which validates the experiment. 3.3. In vivo test Prepatent period increased on infected and treated mice with nanospheres containing nerolidol, and on infected and treated mice with D.A., compared to infected and untreated mice (positive control) (Table 2). Longevity of the group A was of 60 days (exactly the number of days that the experiment lasted). Longevity of the groups A, B, C, D, and E were 60, 4.6, 20.3, 53.8, and 40.2 days, respectively. Mice from the group C had increase longevity, but did not showed curative effect. However, mice from the group D (treated with nanospheres containing nerolidol) had increased longevity and showed 66.66% of curative effect compared to D.A. that showed 33.33%. 3.4. Cytotoxicity assay As shown in Table 3, both treatments did not cause cytotoxicity
Fig. 1. Transmission electron micrographs (TEM) of nanospheres containing nerolidol at 50,000 magnification.
Fig. 2. In vitro activity of different concentrations of nerolidol [a] and nanospheres containing nerolidol [b] against Trypanosoma evansi. Results within a circle were not statistically different (p > 0.05), at the same time (h).
at the concentrations and conditions tested after 24, 48 and 72 h of incubation in PBMC culture (p > 0.05). 4. Discussion While studying new therapeutic options for trypanosomosis, research has revealed that bioactive compounds present in essential oils or plants showed promising results. In addition, treatments more effective and less toxic than conventional drugs have motivated this study. Nanospheres containing nerolidol was able to increase longevity and therapeutic effectiveness compared to positive controls, as well as nerolidol in its free form. There are many ongoing studies using nanotechnology, especially on the development of drugs to treat parasitic infections, and nanospheres have been suggested as effective carries of antiprotozoal drugs (Molina et al., 2001). The results of nanospheres characterization indicate an adequate homogeneity, because all formulations must be monodisperse (PDI < 0.25) with diameter smaller than 300 nm. Moreover, the result of zeta potential value is satisfactory, since the nanostructure shows a negative charge, the system stability tend to be superior with a lower probability for particles aggregation (Friedrich et al., 2008). In this study, we have not observed a good similarity regarding size of nanospheres when compared Zeta-Sizer and TEM analysis. According to Hoffmann et al. (1997), differences in particles sizes depend on the method used, since them TEM provides an image of the insulated middle particle, while spectroscopy photon correlation enables the determination of the radius hydrodynamic in suspended particles. This is the first study to investigate the performance of nanospheres containing nerolidol against T. evansi. Nerolidol and nanospheres had apparently a faster trypanocidal effect than the
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Table 2 Mean and standard deviation of prepatent period, longevity, and mortality for treatments with nerolidol and nanospheres containing nerolidol, and diminazene aceturate (D.A.) in mice experimentally infected by T. evansi. Groups (n ¼ 6)
Treatment
Prepatent period (Day)
Longevity (Day)
Mortality (n)
Therapeutic success (%)
A B C D E
Negative control Positive control Nerolidol (1.0 mL kg1) Nanospheres (1.0 mL kg1) D.A. (3.5 mg kg1)
e 1.0b 1.5b 9.7a 10.9a
60.0a 4.6d 20.3c 53.8a 40.2b
0/6 6/6 6/6 2/6 4/6
e e 0 66.66 33.33
(±0.0) (±0.5) (±2.5) (±2.7)
(±0.0) (±0.5) (±3.2) (±5.6) (±8.0)
Mean followed by the same letter in the same column do not differ significantly according to the Bonferroni's post hoc test. The experiment lasted 60 days after infection.
Table 3 Cytotoxicity on PBMC of nerolidol and nanospheres containing nerolidol. These cells were seeded at 2 105/well in 96-well microplates and incubated for 24, 48, and 72 h in the presence of the compounds at concentrations of 0.5, 1.0 and 2.0%. Viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the optical density was determined at 560 nm. Data are shown as mean of cell viability. (%) ± standard deviation. Compound
Control Nerolidol
Nanospheres
p value
Concentration (%)
PBMC 24 h
48 h
0,5 1,0 2,0 0,5 1,0 2,0
100 ± 0a 99 ± 8a 92 ± 0a 92 ± 14a 107 ± 14a 103 ± 3a 95 ± 13a 0.112
100 ± 110 ± 104 ± 112 ± 105 ± 104 ± 123 ± 0.098
72 h 0a 4a 2a 1a 7a 7a 11a
100 ± 0a 103 ± 7a 103 ± 1z 93 ± 5a 114 ± 8a 110 ± 7a 99 ± 11a 0.121
Means followed by the same letter in the same column do not differ significantly according to Bonferroni's post hoc test.
diminazene aceturate in vitro. Against Microsporum gypseum, nerolidol showed an antifungal effect in vitro with minimum inhibitory concentration of 0.5e2% (Lee et al., 2007). A similarly result was found against important bacterial pathogens, such as Staphylococcus aureus and Escherichia coli, that showed better activity than ciprofloxacin, clindamycin, and tetracycline, standard drugs used to treat these bacterial infections. The mechanism responsible for trypanocidal action is not clearly understood, but against bacteria, nerolidol shows an affinity for biological membranes, where its accumulation may impact on the structural and functional properties of these membranes (Sikkema et al., 1995; Brehm-Stecher and Johnson, 2003). Based on these previous promising in vitro results, we have designed an in vivo experiment using mice infected by T. evansi as the experimental model. For this study, it was used the protocol stablished by Mohd-Shukri and Zainal-Abidin (2011), demonstrated that nerolidol increased longevity of animals infected by T. evansi, but not showed therapeutic efficacy. Same result was observed in this present study, where nerolidol increased longevity compared to positive controls, however these animals showed 100% mortality, corroborating the data described in the literature. The trypanocidal action of nerolidol is based on the ability to inhibit the synthesis of peptidoglycan, which is critically needed for the single-cell bi-layer cell membrane of parasites, such as T. evansi (Ogunlana et al., 1987). Also, the capacity of nerolidol to modify the hydrophobic characteristic on the surface of the cell membrane might lead to changes on the original cell structure or morphology (Turi et al., 1997). Recently, AbouLaila et al. (2010) demonstrated that nerolidol showed inhibitor effects against Babesia sp. due to the capacity to inhibit isoprenoid biosynthesis by inhibiting the incorporation of melavonic acid or acetic acid precursors into dolichol, ergosterol, and ubiquinone. Polymeric nanospheres constitute the most thoroughly investigated nanocarriers for drug delivery (Xu et al., 2007). Due to their polymeric nature, nanospheres generally possess high stability in
biological fluids, such as blood, which qualifies them as promising drug delivery systems against trypanosomes, as well observed by Santos-Magalh~ aes and Mosqueira (2010) against malaria infection. We have decided to develop a nanoformulation that was able to enhance trypanocidal action of nerolidol, and thus, protecting the compound from the acid pH of the stomach, once treatment occurs orally, and some compounds can lose their activity (Shukla et al., 2012). To protect doxorubicin against acidic pH stomach, Shukla et al. (2012) developed nanospheres against leishmaniasis, caused by Leishmania infantum and Leishmania. donavani protozoan. This author demonstrated that nanospheres was able to reduce toxicity and protects the doxorubicin from the stomach, leading to increased efficiency of doxorubicin when compared to its free form. Thus, we believe that polymeric nanospheres can protect the active principle against acidic pH, improving the trypanocidal activity, such as observed in this study. Moreover, nanospheres properties observed in other studies, such as improvement of the therapeutic index, control release, mucosal protection, and its stability in the blood for a long time (Soppimath et al., 2001), might be important therapeutic features responsible for improved nerolidol activity against T. evansi. In vitro, a similarly result was showed between nerolidol and nanospheres containing nerolidol, different that occurs in vivo. We believe that this occurs due to the short time of in vitro test, hindering the occurrence controlled release of drug through the polymeric matrix, as well as observed in others in vitro studies with T. evansi (Gresller et al., 2015). Study conducted by Calvo et al. (1996) demonstrated that nanospheres have the characteristic of rapid release of the drug by diffusion through the polymer matrix. Thus, we believe that during the in vitro test the nerolidol has not diffused through the polymeric matrix due to the short time of in vitro test. Also, the nanospheres feature a kinetic liberation in exponential form, probably due drug diffusion through polymeric matrix for body or erosion of polymeric matrix, releasing the drug during in vivo test (Calvo et al., 1996). Due to the need for trypanocidal substances that are more selective for the parasite and less toxic to host cells, it was important to investigate the cytotoxic activity of nerolidol free or nanoencapsulated against mammalian cells. Both formulations did not cause a toxic effect in PBMC after 24, 48, and 72 h post-incubation. Furthermore, many other cytotoxic assays can be conducted using animal cells to determine the safety of a substance, leading to a future treatment. Lymphocyte is a model used in preliminary studies regarding the protective and toxic effects of substances, being a possible indicator of these type of damage to cells in vivo (Lexis et al., 2006). In conclusion, the present work showed that it is possible to produce nanospheres containing nerolidol (3 mg/mL) with adequate physicochemical characteristics for oral administration. The nanoencapsulation of nerolidol showed improved trypanocidal activity and it is effective in the treatment of mice experimentally infected by T. evansi creating a viable alternative to treat affected animals. In summary, nanoencapsulation may be an interesting approach to improve the efficacy of therapeutic drugs.
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Nerolidol nanospheres increases its trypanocidal efficacy against Trypanosoma evansi: New approach against diminazene aceturate resistance and toxicity Matheus D. Baldissera a, *, Thirssa H. Grando a, Carine F. Souza a, Luciana F. Cossetin a, tia Nascimento b, Ana P.T. da Silva c, Daiane F. Dalla Lana d, Michele R. Sagrillo b, Ka Aleksandro S. Da Silva e, Lenita M. Stefani e, Silvia G. Monteiro a, ** a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil rio Franciscano, Santa Maria, RS, Brazil Laboratory of Cell Culture, Centro Universita c rio Franciscano, Santa Maria, RS, Brazil Laboratory of Nanotechnology, Centro Universita d Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grando do Sul (UFRGS), Porto Alegre, RS, Brazil e , SC, Brazil Department of Animal Science, Universidade do Estado de Santa Catarina, Chapeco b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Nerolidol and nanospheres present trypanocidal action in vitro against Trypanosoma evansi. Nerolidol has trypanocidal action, but without curative effectiveness in vivo. Nanoencapsulation increase curative effectiveness in vivo. Nanoencapsulation may be an interesting approach to improve the efficacy of therapeutic drugs.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2016 Received in revised form 6 April 2016 Accepted 19 April 2016 Available online 21 April 2016
The aims of this study were to develop nerolidol-loaded nanospheres, and to evaluate their efficacy in vitro and in vivo against Trypanosoma evansi, as well as to determine their physicochemical properties, morphology, and any possible side effect in vitro against peripheral blood mononuclear cell (PBMC). The nanospheres showed an adequate particle size (149.5 nm), narrow particle distribution (0.117), negative zeta potential (12.8 mV), and pH of 6.84, such as observed by transmission electron microscopy. In vitro, a trypanocidal effect of nerolidol and nanospheres containing nerolidol was observed at 0.5, 1.0, and 2.0%, i.e., both treatments showed a faster trypanocidal effect compared to chemotherapy (diminazene aceturate e D.A.). T. evansi infected mice were used to evaluate the effects of nerolidol-loaded nanospheres regarding pre-patent period, longevity, and therapeutic efficacy. Oral administration of nerolidol-loaded nanospheres at 1.0 mL/kg/day during 10 days increased mice survival (66.66%) compared to 0% and 33.33% of mice survival when treated with nerolidol in its free form and D.A., respectively. Cytotoxic study indicated that both treatments showed no side effects in vitro against PBMC, an important marker used in toxicological surveys. Therefore, nanoencapsulation increased the therapeutic efficacy of nerolidol against T. evansi, and can be used as an alternative treatment for T. evansi infection. © 2016 Elsevier Inc. All rights reserved.
Keywords: Protozoan Sesquiterpenes Transmission electron microscopy Nanomedicine
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M.D. Baldissera), [email protected] (S.G. Monteiro). http://dx.doi.org/10.1016/j.exppara.2016.04.015 0014-4894/© 2016 Elsevier Inc. All rights reserved.
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1. Introduction
2. Materials and methods
Trypanosoma evansi is a protozoan parasite that affects domestic and wild animals (Silva et al., 2002; Desquesnes et al., 2013), and rarely parasitizes humans (Joshi et al., 2005). It is considered the most widely distributed pathogenic African trypanosomes. This parasite causes a disease known as “surra”, or “Mal das Cadeiras” in horses, and it is mechanically transmitted by biting flies (Stomoxis sp., Hematopota sp., and Chrysops sp.). Diminazene aceturate (D.A.) at a single dose of 3.5 mg kg1 is capable to eliminate the parasites in bloodstream a few hours after administration. However, it has no curative efficacy since trypanosomes may pass through the bloodbrain barrier, reaching the central nervous system, as well known area of refuge to T. evansi during the residual period of drug circulation. Diminazene aceturate does not cross the blood-brain barrier in amounts sufficient to eliminate all the parasites (Masocha et al., 2007). Also, therapeutic dose of D.A. usually shows signs of toxicity, especially related to hepatotoxic and nephrotoxic effects, as reported in the literature (Baldissera et al., 2016a, 2016b). Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol), also known as peruviol, is an aliphatic sesquiterpene alcohol present in essential oils of many plants. Many medical benefits of nerolidol have been identified including antioxidant (Nogueira et al., 2013), antinociceptive (Koudou et al., 2005), anti-ulcer (Klopell et al., 2007), anti-tumor (Lapczynski et al., 2008), and anti-bacterial (Lang and Buchbauer, 2012) properties. Regarding the antiparasitic effect of nerolidol, it has shown antileishmanial (Arruda et al., 2005), antimalarial (Lopes et al., 1999), antischistosomal (Silva et al., 2014), babesicide (AbouLaila et al., 2010) and antitrypanosomal (Mohd-Shukri and Zainal-Abidin, 2011) activities. Regarding the antitrypanosomal activity, nerolidol is able to prolong life of mice infected by T. evansi, however, without curative effectiveness. Thus, we intend to use the benefits of nanotechnology to improve its curative effectiveness due capacity of nanotechnology in increase the therapeutic index, control release of the drugs and cross the blood brain barrier (Soppimath et al., 2001), as well as observed in other treatment against T. evansi described below. Also, some pharmaceuticals difficulties of nerolidol has been observed in the literature, such as solubility problems (Nogueira et al., 2013). Considering the low efficacy of drugs available for the treatment of T. evansi infection, as well as their side effects and great resistance developed by protozoan, the nanotechnology associated with vegetal species may lead to the discovery of alternative ways with appropriate efficiency to treat the disease (Alviano and Alviano, 2009). In this context, nanospheres are a specific type of nanoparticle that have been used successfully because it can protect the antiparasitic drugs from gastric inactivation, protect mucosa against drug toxicity, control drug release, improvement of bioavailability, and decrease side effects (Shukla et al., 2012). Different antiparasitic drugs have been used into nanospheres, such as bis-triazole DO870 for Trypanosoma cruzi infection (Molina et al., 2001). Encapsulated vegetal compounds have shown promising results against T. evansi such as curcumin (Gressler et al., 2015), Melaleuca alternifolia, and Achyrocline satureioides essential oils (Baldissera et al., 2014; Do Carmo et al., 2015), compared to the free form. Different antiparasitic drugs have been tested in nanospheres in order to improve their therapeutic index. To enhance the trypanocidal effect of nerolidol, we have decided to develop a nanoformulation and to test it in vitro and in vivo. Therefore, the aims of this study were to develop and characterize nanospheres containing nerolidol, and to evaluate their efficacy in vitro and in vivo against T. evansi.
2.1. Nerolidol Nerolidol (molecular weight: 222.37 g/mol) was purchased from Sigma-Aldrich® and showed 97% of purity. 2.2. Nanospheres preparation Nanospheres suspension (NS) containing nerolidol was prepared according to the interfacial deposition of preformed polymer (Fessi et al., 1989). The components of the organic phase and aqueous phase (Table 1) were placed in a beaker and kept under magnetic stirring in a water bath at 40 C for 1 h, and the active principle (AP) was added to the organic phase at the end of the heating period. With the aid of a funnel, the organic phase was poured into the aqueous phase and stirred for 10 min. This mixture was concentrated to a final volume of 100 mL in a rotary evaporator to eliminate the organic solvent, adjusting the final concentration of AP to 3 mg mL1 from pure nerolidol (100% concentration). 2.3. Nanospheres characterization The pH determination for the nanosphere suspension was performed using a potentiometer (Digimed®) previously calibrated with buffer solutions at pH 4.0 and 7.0. The determination of the diameter and polydispersity of nanoparticles in suspension were performed by dynamic light scattering using the Zetasizer®, NanoZS from Malvern equipment. The suspensions were diluted 500 times (v/v) in Milli-Q water and the results were determined by the average of three replicates. The zeta potential was obtained using the technique of electrophoretic mobility Zetasizer®, Malvern Nano-ZS instrument. The samples were pre-diluted at 500 times (v/ v) in 10 mM sodium chloride and filtered through a membrane with 0.45 mm. The results were expressed in millivolts (mV). 2.4. Scanning electron microscopy Morphological analysis was determined by transmission electron microscopy (TEM) (JEM 2010, Tokyo, Japan) at 200 KV in operation. For this, the suspension was diluted 10 in ultrapure water, placed on a specimen grid and negatively stained with uranyl acetate (2% W/V). 2.5. Trypanosoma evansi isolate This study was conducted in two consecutive experiments (in vitro and in vivo). The cryopreserved isolate of T. evansi used in these experiments was obtained from a naturally infected dog (Colpo et al., 2005). Two rats (Ra and Rb) were infected Table 1 Composition nerolidol.
of
nanospheres
suspensions
containing
Compounds mL Organic phase Eudragit RS 100® Sorbitan monostereate Nerolidol Acetone Aqueous phase Polysorbate 80 Milli-Q water
1.0 0.766 3.102 267.0 0.766 533.0
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intraperitoneally with blood contaminated by trypomastigotes, which was kept cryopreserved in liquid nitrogen. This process was performed in order to obtain a large amount of viable parasites for in vitro tests (Ra), and to infect the mice in the experimental groups (Rb). 2.6. In vitro test The culture medium for T. evansi was adapted from Baltz et al. (1985) as previously published by Baldissera et al. (2013). The protozoa were acquired from Ra three days post-infection, the rat showed high parasitemia (6.0 105 trypanosomes/mL) and it was anesthetized with isoflurane for blood collection by cardiac puncture. The blood was stored in tubes containing EDTA (ethylenediamine tetraacetic acid). For trypanosome separation, each 200 mL of blood was diluted in complete culture medium (DMEM) 1:1 (v/v), added to microcentrifuge tubes, and centrifuged at 400 g for 10 min at 25 C. The supernatant was removed and resuspended in culture medium and the number of parasites was counted in a Neubauer chamber. The culture medium containing the parasites was distributed into microtiter plates (270 mL/well), and 25 mL of each treatment was added (diluted in culture medium). For this assessment, the nerolidol and nanospheres containing nerolidol were used at concentrations of 0.5, 1.0 and 2.0%. A positive control (0.5% D.A.) was also adopted at the same volume (25 mL). Each well received 20 mL to count the number of parasites at 1, 3, 6 and 9 h after the onset of the experiment in Neubauer chambers. 2.7. Tests in vivo 2.7.1. Animal model Thirty 60-day-old female mice, heterogenic, outbred strain, weighing an average of 30 ± 0.4 g were used as the experimental model. The animals were maintained under controlled light and environment (12:12 h light-dark cycle, 23 ± 1 C, 70% relative humidity) with free access to commercial food and water. Experiments were carried out between 8 a.m. and 5 p.m. All animals were subjected to a period of 30 days for adaptation. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. These procedures were approved by the Animal Welfare Committee of Federal University of Santa Maria under protocol number 8658110915. 2.7.2. Experimental design and parasitemia estimation The mice were assigned to five groups (A - E), with six animals each. The group A consisted of uninfected and untreated animals (negative control); the group B: infected and untreated mice (positive control); the group C: animals infected and treated nerolidol 1.0 mL kg1; the group D: animals infected and treated with nanospheres containing nerolidol 1.0 mL kg1; the group E: animals infected and treated with D.A. in a single dose of 3.5 mg kg1, intramuscularly. Animals from groups B, C, D, and E were inoculated intraperitoneally with 0.06 mL of blood from Rb containing 2.0 105 trypanosomes. A daily supply of nerolidol and nanospheres containing nerolidol was maintained for five days before infection and five days after infection via oral gavage, according to the protocol used by Mohd-Shukri and Zainal-Abidin (2011). One hour post-infection, D.A. was administered according to manufactures recommendation (single dose of 3.5 mg kg1, intramuscularly). The peripheral blood from the tail of the rats was examined daily for parasitemia degree scoring system. Each slide was prepared with fresh blood collected from the tail coccygeal vein, stained by the Romanowski method, and visualized at a
magnification of 1000 according to the method described by Da Silva et al. (2006). The mice were observed for up to 60 days. 2.7.3. Treatment efficacy The number of mice that did not show clinical signs of T. evansi infection and did not die after treatment was used to determine treatment efficacy. Prepatent period, and longevity were also observed. 2.8. Cytotoxicity assay Although the natural products may be efficient, the toxicity of preparations used is usually unknown. However, toxicology tests showed that many plants currently used are highly toxic (Atsamo et al., 2011). Therefore, we decided to determine nerolidol safety by using peripheral blood mononuclear cells (PBMC) culture, once this cellular type is an indicator in vivo of damage to cells (Lexis et al., 2006). 2.8.1. PBMC cells Samples of peripheral blood were supplied by the Clinical Analysis Laboratory of the Franciscan University Center and the project was approved by this same center (CAAE: 31211214.4.0000.5306). Peripheral blood samples were collected from three apparently healthy volunteers (22e25 years old), who did not smoke, neither drank alcoholic beverages more than two times a week, nor took prescription drugs and without autoimmune or infectious diseases. The samples were collected after 12 h of overnight fasting by venipuncture using a top Vacutainer® (BD Diagnostics, Plymouth, UK) and heparin tubes. PBMC were separated by Histopaque-1077 (SigmaeAldrich Co., St Louis, USA) by density gradient centrifugation using 4 mL of blood samples. After further centrifugation for 15 min at 2.500 g, cells were transferred to culture media containing 5 mL of RPMI 1640 supplemented with 10% fetal bovine serum, and 1% of penicillin and streptomycin. The cells were cultured in a 96-well microplate at an initial density of 2 105 for 24, 48 and 72 h at 37 C in a 5% humidified CO2 atmosphere (Wilms et al., 2005). 2.8.2. Cell viability The cytotoxicity of the nerolidol and nanospheres containing nerolidol against PBMC was evaluated as described by Sagrillo et al. (2015), using the tetrazolium salt MTT 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric method based on the cleavage of the reagent by dehydrogenases in viable cells (Mosmann, 1983). In the microplates, cells were incubated at 37 C in a humid atmosphere with 5% CO2 for 24, 48, and 72 h. At the end of the incubation, 50 mL of MTT solution was added to each well of the microplate and the cells were incubated for three more hours. The supernatant was removed and 200 mL of dimethyl sulfoxide (DMSO) were added to each well. The microplate was analyzed using an ELISA reader at a wavelength of 560 nm. As a control, cells were grown in a medium lacking the constituents. The experiment was performed in triplicate. Finally, cell viability was determined, which was expressed as a percentage of the control value. 2.9. Statistical analysis The data met the assumption of parametric testing according to the Kolmogorov-Smirnov test. The bilateral two-way analysis of variance (ANOVA) followed by the Bonferroni's post hoc test were used for comparison of means. Differences between groups were rated significant at p < 0.05. All analysis were carried out in an IBM compatible computer using the Statistical Package for the Social Sciences (SPSS) software 20. Results were presented as
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mean ± standard deviation. 3. Results 3.1. Characterization of nanospheres Composition of nanospheres suspensions containing nerolidol was showed in Table 1. The nerolidol nanospheres were evaluated regarding their physical and chemical properties. The droplet size was around 149.5 nm, polydispersity of 0.117, the potential zeta was 12.8 mV and the hydrogen potential (pH) was around 6.84. TEM image show the morphology spherical of the nanospheres (Fig. 1). The mean particle size observed by TEM analysis was in agreement with those obtained by spectroscopy correlation of photons. 3.2. In vitro test The trypanocidal effect of nerolidol (Fig. 2A) and nanospheres containing nerolidol (Fig. 2B) on T. evansi was directly proportional to the concentration used. A reduction of live trypomastigotes was observed at all concentrations compared to the control group. After 6 h, there were no living trypomastigotes in 1% and 2% concentrations using nerolidol and nanospheres containing nerolidol. After 9 h of the beginning of the assay, there were also no living trypomastigotes in 0.5% concentration as well as on the D.A. treatment. On the other hand, parasites were alive in positive control samples, which validates the experiment. 3.3. In vivo test Prepatent period increased on infected and treated mice with nanospheres containing nerolidol, and on infected and treated mice with D.A., compared to infected and untreated mice (positive control) (Table 2). Longevity of the group A was of 60 days (exactly the number of days that the experiment lasted). Longevity of the groups A, B, C, D, and E were 60, 4.6, 20.3, 53.8, and 40.2 days, respectively. Mice from the group C had increase longevity, but did not showed curative effect. However, mice from the group D (treated with nanospheres containing nerolidol) had increased longevity and showed 66.66% of curative effect compared to D.A. that showed 33.33%. 3.4. Cytotoxicity assay As shown in Table 3, both treatments did not cause cytotoxicity
Fig. 1. Transmission electron micrographs (TEM) of nanospheres containing nerolidol at 50,000 magnification.
Fig. 2. In vitro activity of different concentrations of nerolidol [a] and nanospheres containing nerolidol [b] against Trypanosoma evansi. Results within a circle were not statistically different (p > 0.05), at the same time (h).
at the concentrations and conditions tested after 24, 48 and 72 h of incubation in PBMC culture (p > 0.05). 4. Discussion While studying new therapeutic options for trypanosomosis, research has revealed that bioactive compounds present in essential oils or plants showed promising results. In addition, treatments more effective and less toxic than conventional drugs have motivated this study. Nanospheres containing nerolidol was able to increase longevity and therapeutic effectiveness compared to positive controls, as well as nerolidol in its free form. There are many ongoing studies using nanotechnology, especially on the development of drugs to treat parasitic infections, and nanospheres have been suggested as effective carries of antiprotozoal drugs (Molina et al., 2001). The results of nanospheres characterization indicate an adequate homogeneity, because all formulations must be monodisperse (PDI < 0.25) with diameter smaller than 300 nm. Moreover, the result of zeta potential value is satisfactory, since the nanostructure shows a negative charge, the system stability tend to be superior with a lower probability for particles aggregation (Friedrich et al., 2008). In this study, we have not observed a good similarity regarding size of nanospheres when compared Zeta-Sizer and TEM analysis. According to Hoffmann et al. (1997), differences in particles sizes depend on the method used, since them TEM provides an image of the insulated middle particle, while spectroscopy photon correlation enables the determination of the radius hydrodynamic in suspended particles. This is the first study to investigate the performance of nanospheres containing nerolidol against T. evansi. Nerolidol and nanospheres had apparently a faster trypanocidal effect than the
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Table 2 Mean and standard deviation of prepatent period, longevity, and mortality for treatments with nerolidol and nanospheres containing nerolidol, and diminazene aceturate (D.A.) in mice experimentally infected by T. evansi. Groups (n ¼ 6)
Treatment
Prepatent period (Day)
Longevity (Day)
Mortality (n)
Therapeutic success (%)
A B C D E
Negative control Positive control Nerolidol (1.0 mL kg1) Nanospheres (1.0 mL kg1) D.A. (3.5 mg kg1)
e 1.0b 1.5b 9.7a 10.9a
60.0a 4.6d 20.3c 53.8a 40.2b
0/6 6/6 6/6 2/6 4/6
e e 0 66.66 33.33
(±0.0) (±0.5) (±2.5) (±2.7)
(±0.0) (±0.5) (±3.2) (±5.6) (±8.0)
Mean followed by the same letter in the same column do not differ significantly according to the Bonferroni's post hoc test. The experiment lasted 60 days after infection.
Table 3 Cytotoxicity on PBMC of nerolidol and nanospheres containing nerolidol. These cells were seeded at 2 105/well in 96-well microplates and incubated for 24, 48, and 72 h in the presence of the compounds at concentrations of 0.5, 1.0 and 2.0%. Viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the optical density was determined at 560 nm. Data are shown as mean of cell viability. (%) ± standard deviation. Compound
Control Nerolidol
Nanospheres
p value
Concentration (%)
PBMC 24 h
48 h
0,5 1,0 2,0 0,5 1,0 2,0
100 ± 0a 99 ± 8a 92 ± 0a 92 ± 14a 107 ± 14a 103 ± 3a 95 ± 13a 0.112
100 ± 110 ± 104 ± 112 ± 105 ± 104 ± 123 ± 0.098
72 h 0a 4a 2a 1a 7a 7a 11a
100 ± 0a 103 ± 7a 103 ± 1z 93 ± 5a 114 ± 8a 110 ± 7a 99 ± 11a 0.121
Means followed by the same letter in the same column do not differ significantly according to Bonferroni's post hoc test.
diminazene aceturate in vitro. Against Microsporum gypseum, nerolidol showed an antifungal effect in vitro with minimum inhibitory concentration of 0.5e2% (Lee et al., 2007). A similarly result was found against important bacterial pathogens, such as Staphylococcus aureus and Escherichia coli, that showed better activity than ciprofloxacin, clindamycin, and tetracycline, standard drugs used to treat these bacterial infections. The mechanism responsible for trypanocidal action is not clearly understood, but against bacteria, nerolidol shows an affinity for biological membranes, where its accumulation may impact on the structural and functional properties of these membranes (Sikkema et al., 1995; Brehm-Stecher and Johnson, 2003). Based on these previous promising in vitro results, we have designed an in vivo experiment using mice infected by T. evansi as the experimental model. For this study, it was used the protocol stablished by Mohd-Shukri and Zainal-Abidin (2011), demonstrated that nerolidol increased longevity of animals infected by T. evansi, but not showed therapeutic efficacy. Same result was observed in this present study, where nerolidol increased longevity compared to positive controls, however these animals showed 100% mortality, corroborating the data described in the literature. The trypanocidal action of nerolidol is based on the ability to inhibit the synthesis of peptidoglycan, which is critically needed for the single-cell bi-layer cell membrane of parasites, such as T. evansi (Ogunlana et al., 1987). Also, the capacity of nerolidol to modify the hydrophobic characteristic on the surface of the cell membrane might lead to changes on the original cell structure or morphology (Turi et al., 1997). Recently, AbouLaila et al. (2010) demonstrated that nerolidol showed inhibitor effects against Babesia sp. due to the capacity to inhibit isoprenoid biosynthesis by inhibiting the incorporation of melavonic acid or acetic acid precursors into dolichol, ergosterol, and ubiquinone. Polymeric nanospheres constitute the most thoroughly investigated nanocarriers for drug delivery (Xu et al., 2007). Due to their polymeric nature, nanospheres generally possess high stability in
biological fluids, such as blood, which qualifies them as promising drug delivery systems against trypanosomes, as well observed by Santos-Magalh~ aes and Mosqueira (2010) against malaria infection. We have decided to develop a nanoformulation that was able to enhance trypanocidal action of nerolidol, and thus, protecting the compound from the acid pH of the stomach, once treatment occurs orally, and some compounds can lose their activity (Shukla et al., 2012). To protect doxorubicin against acidic pH stomach, Shukla et al. (2012) developed nanospheres against leishmaniasis, caused by Leishmania infantum and Leishmania. donavani protozoan. This author demonstrated that nanospheres was able to reduce toxicity and protects the doxorubicin from the stomach, leading to increased efficiency of doxorubicin when compared to its free form. Thus, we believe that polymeric nanospheres can protect the active principle against acidic pH, improving the trypanocidal activity, such as observed in this study. Moreover, nanospheres properties observed in other studies, such as improvement of the therapeutic index, control release, mucosal protection, and its stability in the blood for a long time (Soppimath et al., 2001), might be important therapeutic features responsible for improved nerolidol activity against T. evansi. In vitro, a similarly result was showed between nerolidol and nanospheres containing nerolidol, different that occurs in vivo. We believe that this occurs due to the short time of in vitro test, hindering the occurrence controlled release of drug through the polymeric matrix, as well as observed in others in vitro studies with T. evansi (Gresller et al., 2015). Study conducted by Calvo et al. (1996) demonstrated that nanospheres have the characteristic of rapid release of the drug by diffusion through the polymer matrix. Thus, we believe that during the in vitro test the nerolidol has not diffused through the polymeric matrix due to the short time of in vitro test. Also, the nanospheres feature a kinetic liberation in exponential form, probably due drug diffusion through polymeric matrix for body or erosion of polymeric matrix, releasing the drug during in vivo test (Calvo et al., 1996). Due to the need for trypanocidal substances that are more selective for the parasite and less toxic to host cells, it was important to investigate the cytotoxic activity of nerolidol free or nanoencapsulated against mammalian cells. Both formulations did not cause a toxic effect in PBMC after 24, 48, and 72 h post-incubation. Furthermore, many other cytotoxic assays can be conducted using animal cells to determine the safety of a substance, leading to a future treatment. Lymphocyte is a model used in preliminary studies regarding the protective and toxic effects of substances, being a possible indicator of these type of damage to cells in vivo (Lexis et al., 2006). In conclusion, the present work showed that it is possible to produce nanospheres containing nerolidol (3 mg/mL) with adequate physicochemical characteristics for oral administration. The nanoencapsulation of nerolidol showed improved trypanocidal activity and it is effective in the treatment of mice experimentally infected by T. evansi creating a viable alternative to treat affected animals. In summary, nanoencapsulation may be an interesting approach to improve the efficacy of therapeutic drugs.
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