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A nanotechnology based new approach for Trypanosoma evansi chemotherapy: In vitro and vivo trypanocidal effect of (-)-α-bisabolol
Accepted Manuscript A nanotechnology based new approach for Trypanosoma evansi chemotherapy: In vitro and vivo trypanocidal effect of (-)-α-bisabolol Matheus D. Baldissera, Thirssa H. Grando, Carine F. de Souza, Luciana F. Cossetin, Ana P.T. da Silva, Janince L. Giongo, Silvia G. Monteiro PII:
S0014-4894(16)30213-2
DOI:
10.1016/j.exppara.2016.09.018
Reference:
YEXPR 7309
To appear in:
Experimental Parasitology
Received Date: 24 July 2016 Revised Date:
15 September 2016
Accepted Date: 27 September 2016
Please cite this article as: Baldissera, M.D., Grando, T.H., de Souza, C.F., Cossetin, L.F., da Silva, A.P.T., Giongo, J.L., Monteiro, S.G., A nanotechnology based new approach for Trypanosoma evansi chemotherapy: In vitro and vivo trypanocidal effect of (-)-α-bisabolol, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.09.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A nanotechnology based new approach for Trypanosoma evansi chemotherapy: in
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vitro and vivo trypanocidal effect of (-)-α-Bisabolol
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Matheus D. Baldisseraa*, Thirssa H. Grandoa, Carine F. de Souzab, Luciana F. Cossetina,
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Ana P.T. da Silvac, Janince L. Giongod, Silvia G. Monteiroa*.
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(UFSM), Santa Maria, RS, Brazil.
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b
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Department of Microbiology and Parasitology, Universidade Federal de Santa Maria
Department of Physiology and Pharmacology, Universidade Federal de Santa Maria
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(UFSM), Santa Maria, RS, Brazil.
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Brazil
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Uruguai e das Missões/URI, Santiago, RS, Brazil.
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Laboratory of Pharmaceutical Technology, Universidade Regional Integrada do Alto
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Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, RS,
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*Author for correspondence: [email protected] (M.D. Baldissera) and
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[email protected] (S.G. Monteiro).
ACCEPTED MANUSCRIPT ABSTRACT
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The aim of this study was to evaluate the in vitro and in vivo susceptibility of
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Trypanosoma evansi to α-Bisabolol and solid lipid nanoparticles containing α-Bisabolol
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(SLN-B). In vitro, a trypanocidal effect of α-Bisabolol and SLN-B was observed when
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used at 0.5, 1 and 2 % concentrations, i.e., the concentrations of 1 and 2 % showed a
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faster trypanocidal effect when compared to chemotherapy (diminazene aceturate –
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D.A.). T. evansi infected mice were treated with α-Bisabolol and SLN-B at a dose of 1.0
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mL kg-1 during seven days via oral gavage. In vivo, treatment with SLN-B, D.A. and
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D.A. associated with SLN-B were able to increase (p<0.05) the pre-patent period and
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longevity when compared to positive control (infected and untreated animals), but
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showed no curative efficacy. T. evansi infected mice treated with D.A. associate with
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SLN-B, where a curative efficacy of 50 % was found, a much better result when D.A
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and SLN-B were used alone (16.66 %). In summary, the association with D.A + SLN-B
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can be used as an alternative to improve the therapeutic effectiveness of D.A., and for
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treatment of infected animals with T. evansi. Also, the nanotechnology associated with
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natural products arises an important alternative for the improve the trypanocidal action.
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Keywords: “surra”; nanotechnology; alternative treatment.
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1 INTRODUCTION
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Trypanosoma evansi is the most widely distributed of the pathogenic African
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trypanosomes, affecting domestic and wild animals (Silva et al., 2002; Desquesnes et
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al., 2013), but rarely parasitizes humans (Joshi et al., 2005). The parasite causes a
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disease known as “surra”, and it is mechanically transmitted by biting flies (Stomoxis
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sp., Hematopota sp., and Chrysops sp.), and the principal host species varies according
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to geographical location, parasitizing principally horses, camels, cattle, dogs and cats
ACCEPTED MANUSCRIPT (Desquesnes et al., 2013). Diminazene aceturate (D.A.) at a single dose of 3.5 mg kg-1 is
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capable to providing an elimination of parasites in bloodstream a few hours after
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administration, however, it has no curative efficacy because relapses parasitemia
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occurring due trypanosomes pass through the blood-brain barrier, finding allocated in
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central nervous system, a refuge area for T. evansi during the residual period of the drug
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circulation. Diminazene aceturate does not cross the blood-brain barrier in amounts
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sufficient to eliminate all the parasites (Masoha et al., 2007). Also, therapeutic dose of
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D.A. usually shows signs of toxicity, especially related to hepatotoxic and nephrotoxic
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effects, as reported in the literature (Baldissera et al., 2016a; Baldissera et al., 2016b).
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α-Bisabolol ((-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)5-heptein-2-ol)), also
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known as levomenol, is an sesquiterpene alcohol present in essential oils of several
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plants. With respect to the antiparasitic effect of α-bisabolol, it has shown strong
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antileishmanial activity (Morales-Yuste et al., 2010; Rottini et al., 2015, Corpas-López
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et al., 2015). Regarding the leishmanicidal activity, α-bisabolol has been considered a
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promising compound for the treatment of visceral leishmaniasis, that was able to
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eliminate the promastigotes forms of Leishmania infantum and Leishmania amazonensis
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(Rottini et al., 2015).
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Nanomedicine have been studied for treatment of parasite infections because
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they are able to deliver the drug to the specific target in human and animals body where
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the parasite is located, such as blood and tissues (Mosqueira et al., 2004; Branquinho et
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al., 2014; Morilla and Romero, 2015). Solid lipid nanoparticles (SLN) are a specific
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type of nanoparticle that have been used successfully because protect the drugs from
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chemical degradation, possibility of sustained drug release and enhanced drug
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solubility, (Souto 2009; Souto and Muller 2010). Different antiparasitic drug have been
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loaded into SLN for improving the therapeutic efficacy, such as curcuminoids for
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malaria treatment (Nayak et al., 2010). According to Watkins et al. (2015) the utilization of nanotechnology with
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natural compounds is a rapidly developing field due advantages to the delivery in the
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treatment of parasites diseases. The incorporation of nanoparticles can increase the
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bioavailability, targeting, and controlled-release profiles of the natural products.
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Example of this is the encapsulation of lactone, a natural compound, against Chagas
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disease (Branquinho et al., 2014). Against T. evansi infection, natural products
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encapsulated has established promising results such use of curcumin (Gresller et al.,
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2015), and Melaleuca alternifolia and Achyrocline satureioides essential oils
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(Baldissera et al., 2014, Do Carmo et al., 2015), when compared to the free form.
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Based in this evidences, the aims of this study was to develop and characterize
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solid lipid nanoparticles containing α-bisabolol (SLN-B), and to evaluate their efficacy
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in vitro and in vivo against T. evansi.
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2 MATERIALS AND METHODS
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2.1. (-)-α-Bisabolol
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2.2. Preparation of SLN-B
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(-)-α-Bisabolol (molecular weight: 222.37 g/mol) was purchased from Sigma-
Aldrich® and exhibit 93 % of purity.
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The suspension of SLN-B was prepared according the method described by
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Raffin et al. (2012). The components of the SLN-B are described in Table 1. The
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components of the lipid phase and aqueous phase were separately placed in a beaker and
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and the active principle (AP) was added to the lipid phase at the end of the heating
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period. With the aid of a funnel, the aqueous phase was poured into the lipid phase and
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stirring is continued for 10 minutes. The dispersion of nanoparticles was performed
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using the Ultra-Turrax® T25 during 20 min at 20.000 rpm.
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2.3. Characterization of SLN-B
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The pH determination was performed directly in the suspensions in
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potentiometer (Digimed®) previously calibrated with buffer solutions pH 4.0 and 7.0.
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The determination of the diameter and polydispersity of nanoparticles in suspension
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were performed by dynamic light scattering, the Zetasizer®, Nano-ZS from Malvern
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equipment. The suspensions were diluted 500 times (v:v) in Milli-Q water and the
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results were determined by the average of three replicates. The zeta potential of the
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nanospheres suspensions were obtained using the technique of electrophoretic mobility
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Zetasizer®, Malvern Nano-ZS instrument. The samples were pre-diluted at 500 times
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(v:v) in 10 mM sodium chloride and filtered through a membrane with 0.45
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micrometers. The results are expressed in millivolts (mV) from a mean of three
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determinations.
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2.4. Trypanosoma evansi isolate This study was conducted in two consecutive experiments (in vitro and in vivo).
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The cryopreserved isolate of T. evansi used in these experiments was obtained from a
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naturally infected dog (Colpo et al. 2005). Two rats (Ra and Rb) were infected
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intraperitoneally with blood contaminated by trypomastigotes, which was kept
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cryopreserved in liquid nitrogen. This process was performed in order to obtain a large
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amount of viable parasites for in vitro tests (Ra), and to infect the mice in the
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experimental groups (Rb).
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2.5. In vitro test The culture medium for T. evansi was adapted from Baltz et al. (1985) as
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previously published by Baldissera et al. (2013). The protozoans were acquired from the
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rat infected with the T. evansi isolate (Ra). At three days post-infection, the rat showed
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high parasitemia (3.0 x 104 trypanosomes/µL) and it was anesthetized with isoflurane
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for blood collection by cardiac puncture. The blood was stored in tubes containing
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EDTA (ethylenediamine tetraacetic acid). For trypanosomes separation, each 200 µL of
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blood was diluted in complete culture medium (DMEM) 1:1 (v/v), added to
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microcentrifuge tubes and centrifuged at 400 x g for 10 minutes at 25 °C. The
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supernatant was removed and resuspended in culture medium and the number of
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parasites was counted in a Neubauer chamber.
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The culture medium containing the parasites was distributed in microtiter plates
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(270 µL/well), and 25 µL of each treatment were added (diluted in culture medium). For
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this assessment, the α-bisabolol and SLN-B were used at concentrations of 0.5, 1.0 and
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2.0%. A positive control (0.5% D.A.) was also adopted at the same volume (25 µL). 20
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µL of each well was used to the count the number of parasites at 1, 3, 6 and 9 hours
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after the onset of the experiment in the Neubauer chambers. The in vitro tests were
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performed in duplicate.
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2.6. In vivo test
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2.6.1. Animal model
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weighing an average of 28 ± 0.5 g were used as the experimental model. The animals
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were maintained under controlled light and environment (12:12 h light-dark cycle, 22 ±
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1 °C, 70% relative humidity) with free access to commercial food and water.
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Experiments were carried out between 8 am and 5 pm. All animals were subjected to a
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period of 15 days for adaptation. All efforts were made to minimize animal suffering
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and to reduce the number of animals used in the experiments.
2.6.2. Experimental design and parasitemia estimation
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The mice were assigned to six groups (A - F), with six animals each. Group A
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consisted of uninfected and untreated animals (negative control); Group B: infected and
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untreated mice (positive control); Group C: animals infected and treated α-bisabolol 1.0
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mL kg-1; Group D: animals infected and treated with SLN-B 1.0 mL kg-1; Group E:
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animals infected and treated with D.A; Group F: infected and treated with combination
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of D.A. (3.5 mg kg-1 – intramuscularly) and SLN-B (1.0 mL kg-1 – orally). The infected
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animal groups were inoculated intraperitoneally with 0.05 mL of blood from Rb
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containing 2.0 x 105 trypanosomes.
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The D.A. was intramuscularly injected in a single dose of 3.5 mg kg-1, according
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the manufactures recommendation. At one hour post-infection, α-bisabolol and SLN-B
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were orally administered. A daily supply of α-bisabolol and SLN-B was maintained for
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seven days according the protocol used by Corpas-López et al. (2015).
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The peripheral blood from the tail of the rats was examined daily for scoring the
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degree of parasitemia. Each slide was prepared with fresh blood collected from the tail
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coccygeal vein, stained by the Romanowski method, and visualized at a magnification
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of 1,000 x according to the method described by Da Silva et al. (2006). The mice were
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observed for up to 60 days.
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2.6.3. Treatment efficacy The number of mice that did not show clinical signs of T. evansi infection and
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did not die after treatment determined the treatment efficacy. Prepatent period,
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longevity and animal mortality were also observed.
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2.7. Statistical analysis
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The data met the assumption of parametric testing according to the Kolmogorov-
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Smirnov test. The bilateral two-way analysis of variance (ANOVA) followed by the
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Bonferroni post-hoc test were used for comparison of means. Differences between
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groups were rated significant at p<0.05. All analysis were carried out in an IBM
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compatible computer using the Statistical Package for the Social Sciences (SPSS)
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software 20. Results were presented as means ± standard deviation.
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3 RESULTS
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3.1. Characterization of SLN-B
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SLN-B was evaluated regarding their physical and chemical properties (Table
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2). The particle size was around 191.8 nanometers, and the polydispersion index was
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0.317, with a zeta potential of – 7.76 mV and pH 6.83.
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3.2. In vitro test
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proportional to the concentration used (Figure 1). A reduction of live trypomastigotes
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was observed at all concentrations when compared to the control group. After 6h, there
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were no living trypomastigotes in 1 % and 2 % concentrations using α-bisabolol. After
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9 h of assay, there were also no living trypomastigotes in 0.5 % concentration as well as
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on the D.A. treatment (Figure 1-A)
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The SLN-B are shown in Figure 1-B. Similarly, a reduction of live
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trypomastigotes was observed at all concentrations when compared to the control group.
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After 6h, there were no living trypomastigotes in 1 % and 2 % concentrations. After 9 h
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of assay, there were also no living trypomastigotes in 0.5 % concentration as well as on
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the D.A. treatment. A contrary, in control samples the parasites were all alive, what
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validates our experiment.
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3.3. In vivo test
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Longevity of the group A exactly represented by the days that the experiment
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lasted (60 days) (Table 3). Longevity in the groups B, C, D, E and F were 4.5, 5.0, 32.3,
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30.2 and 48.2 days, respectively. The groups D (SLN-B), E (D.A.) and F (D.A + SLN-
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B) had no curative efficacy, but increased longevity compared to the group B (positive
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control) (p<0.05). The group F showed an increase (p<0.05) longevity when compared
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the treatment with SLN-B and D.A. isolated, and showed 50 % of curative
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effectiveness, while the groups D and E showed 16.66 % of therapeutic effectiveness.
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Prepatent period increased in the group D, E and F when compared to group B (p<0.05),
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but the prepatent period is higher in the group F compared to groups D and E.
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4. DISCUSSION
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of new drugs, since the chemotherapy of T. evansi infection present serious limitations.
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According Tamma et al. (2012) combination therapy is considered as a new and
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promising tool because enhance their parasitic performance and decrease development
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of drug resistance via synergistic or agonistic interactions between used drugs, such as
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observed in this study. In this sense, a combination of D.A. and SLN-B exhibited
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superior activity against T. evansi when compared to the use of D.A. individual.
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On the in vitro assessment, the trypanocidal activity against T. evansi was
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verified to be proportional to the dose applied for α-bisabolol and SLN-B formulations.
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Previous studies suggest that α-bisabolol shows antiparasitic activity in vitro against
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other important blood protozoa, such as L. amazonensis and L. infantum. Recent study
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demonstrated that α-bisabolol showed significant antileishmanial activity against
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promastigotes with a 50 % effective concentration (IC50) of 8.07 µg/mL (24 h) and 4.26
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µg/mL (48 h). Against intracellular amastigotes the IC50 of α-bisabolol was 4.15 µg/mL
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(Rottini et al., 2015). Also, Morales-Yust et al. (2015) demonstrated that 1000 and 500
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µg/mL of α-bisabolol achieved 100 % inhibition of promastigote form of L. infantum in
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vitro. According to recent studies, the alterations in membrane permeability, principally
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in the mitochondrial membrane, may lead to parasite death (Salomão et al., 2013).
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According this author, the possible mechanism action of α-bisabolol is related with the
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capacity to cross the mitochondrial membrane, which can lead to parasite death. Also,
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studies have been demonstrated that sesquiterpenes compounds are capable to inhibit
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the synthesis of ergosterol, an important component of cell membrane. This fact has
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been reported by many authors attempting to explain the antileishmanial action of
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sesquiterpenes compounds extracted from plants (Vendramentto et al., 2010; Medeiros
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et al., 2011; Baldissera et al., 2016c).
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ACCEPTED MANUSCRIPT Based on these previous promising in vitro results, we have designed an in vivo
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experiment using mice infected by T. evansi as the experimental model Although the
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therapeutic protocol used with α-bisabolol and SLN-B did not present curative efficacy,
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an increased in longevity of animals treated with SLN-B was observed. In addition,
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SLN-B showed a similar curative efficacy when compared to D.A. It is important
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emphasize that nanotechnology was able to ameliorate the trypanocidal action of α-
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bisabolol, due the increase of longevity and the presence of efficacy therapeutic (16.66
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%) when compared to free α-bisabolol that showed no curative effectiveness (0%),
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similar results observed by Gressler et al. (2015) using curcumin and Baldissera et al.
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(2016c) using α-terpinene against T. evansi. Recently, study published by Watkins et al.
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(2015) demonstrated that natural product-based on nanotechnology would be a major
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advance in the efforts to increase their therapeutic effects. The main features of SLN are
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physical
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biocompatibility and degradability (Nayak et al., 2010).
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nanoparticles can significantly increase the bioavaibility of natural products in vivo, due
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this capability of manipulate the particles in order to target specific areas of the body
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and control the release of drugs, similar observed by Nayak et al. (2010) using
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curcuminoids against Plasmodium berghei.
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In addition, the association of D.A. with SLN-B increased longevity compared
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to the group B (positive control), and had 50 % of therapeutic efficacy, compared to
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group C (treated only D.A.), with 16.66 % of therapeutic efficacy. Therefore, the D.A or
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SLN-B alone would not be successful in the treatment of trypanosomosis, but when
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using associated, a synergic effect occurs, which could result on a new option for this
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disease treatment. Recently, a study demonstrated that D.A., when associated with other
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terpene compound (α-terpinene), showed an increase of therapeutic efficacy (57.14 %)
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when compared to D.A. alone (14.28 %). α-Bisabolol, in its conventional and nanostructured forms has a trypanocidal
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action in vitro, but only nanostructured leads to higher animals longevity, but it was not
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effective in the treatment of mice experimentally with T. evansi. D.A. when associated
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with SLN-B achieved therapeutic success, becoming an alternative method to treat and
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control infections caused by T. evansi.
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Ethics Committee
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The procedures were approved by the Animal Welfare Committee of Federal University
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of Santa Maria under number 6583091215.
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REFERENCES
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Baldissera, M.D., Da Silva, A.S., Oliveira, C.B., Zimmermann, C.E.P., Vaucher, R.A.,
279
Santos, R.C.V., Rech, V.C., Tonin, A.A., Giongo, J.L., Mattos, C.B., Koester, L.,
280
Santurio, J.M., Monteiro, S.G., 2013. Trypanocidal activity of the essential oils in their
281
conventional and nanoemulsion forms: In vitro tests. Exp. Parasitol.134, 356-361.
EP
282
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277
Baldissera, M.D., Oliveira, C.B., Rech, V.C., Rezer, J.F.P., Sagrillo, M.R., Alves, M.P.,
284
da Silva, A.P.T., Leal, D.B.R., Boligon, A.A., Athayde, M.L., Da Silva, A.S., Mendes,
285
R.E., Monteiro, S.G., 2014. Treatment with essential oil of Achyrocline satureioides in
286
rats infected with Trypanosoma evansi: Relationship between protective effect and
287
tissue damage. Pathol. Res. Pract. 210, 1068-1074.
AC C
283
288 289
Baldissera, M.D., Bottari, N.B., Rech, V.C., Nishihira, V.S.K., Oliveira, C.B., Cargnin,
290
L.P., Moresco, R.N., Thomé, G.R., Schetinger, M.R.C., Morsch, V.M., Monteiro, S.G.,
291
Tonin, A.A., Da Silva, A.S., 2016a. Combination of diminazene aceturate and
292
resveratrol reduces the toxic effects of chemotherapy in treating Trypanosoma evansi
293
infection. Comp. Clin. Pathol. 25, 137-144.
ACCEPTED MANUSCRIPT 294
Baldissera, M.D., Gonçalves, R.A., Sagrillo, M.R., Grando, T.H., Ritter, C.S., Grotto,
296
F.S., Brum, G.F., da Luz, S.C.A., Silveira, S.O., Fausto, V.P., Boligon, A.A., Vaucher,
297
R.A., Stefani, L.M., da Silva, A.S., Souza, C.F., Monteiro, S.G., 2016b. Effects of
298
Treatment with the anti-parasitic drug diminazene aceturate on antioxidant enzymes in
299
rat liver and kidney. Naunyn-Schmiedeberg’s Arch. Pharmacol. 389, 429-438.
RI PT
295
300
Baldissera, M.D., Grando, T.H., Souza, C.F., Gressler, L.T., Stefani, L.M., Da Silva,
302
A.S., Monteiro, S.G., 2016c. In vitro and in vivo action of α-terpinen-4-ol, γ-terpinene
303
and α-terpinene against Trypanosoma evansi. Exp. Parasitol. 162, 43-48.
SC
301
304
Baltz, T., Baltz, D., Giroud, C., Crockett, J., 1985. Cultivation in a semi-defined
306
medium of animals infective forms of Trypanosoma brucei, T. equiperdum, T. evansi,
307
T. rhodesiensi and T. gambiense. The EMBO Journal 4, 1273-1277.
M AN U
305
308
Branquinho, R.T., Mosqueira, V.C.F., de Oliveira-Silva, J.C.V., Simões-Silva, M.R.,
310
Saúde-Guimarães, D.A., de Lana, M., 2014. Sesquiterpene Lactone in Nanostructured
311
parenteral dosage form is efficacious in experimental Chagas disease. Antimicrob.
312
Agents Chemother. 58, 2067-2075.
TE D
309
313
Colpo, C.B., Monteiro, S.G., Stainki, D.R., Colpo, E.T.B., Henriques, G.B., 2005.
315
Natural infection by Trypanosoma evansi in dogs. Cienc. Rural 35, 717-719.
316
EP
314
Corpas-López, V., Morillas-Márquez, F., Navarro-Moll, M.C., Merino-Espinosa, G.,
318
Díaz-Sáez, V., Martín-Sánchez, J., 2015. (-) α-bisabolol, a promising oral compound for
319
the Treatment of visceral leishmaniasis. J. Nat. Prod. 78, 1202-1207.
320
AC C
317
321
Da Silva, A.S., Doyle, R.L., Monteiro, S.G., 2006. Métodos de contenção e confecção
322
de esfregaço sanguíneo para pesquisa de hemoparasitas em ratos e camundongos. Rev.
323
FZVA 13, 83-87.
324 325
Desquesnes, M., Holzmuller, P., Lai, H., Dargantes, A., Lun, Z.L., Jittaplapong, S.,
326
2013. Trypanosoma evansi and Surra: A review and perspectives on origin, history,
ACCEPTED MANUSCRIPT distribution, taxonomy, morphology, hosts and pathogenic effects. BioMed Res. Int.
328
2013, 19416.
329
Do Carmo, G.M., Baldissera, M.D., Vaucher, R.A., Rech, V.C, Oliveira, C.B., Sagrillo,
330
M.R., Boligon, A.A., Athayde, M.L., Alves, M.P., França, R.T., Lopes, S.T.A.,
331
Schwertz, C.I., Mendes, R.E., Monteiro, S.G., Da Silva, A.S., 2015. Effect of the
332
treatment with Achyrocline satureioides (free and nanocapsules essential oil) and
333
diminazene aceturate on hematological and biochemical parameters in rats infected by
334
Trypanosoma evansi. Exp. Parasitol. 149, 39-46
RI PT
327
335
Gressler, L.T., Oliveira, C.B., Coradini, K., Dalla Rosa, L., Grando, T.H., Baldissera,
337
M.D., Zimmermann, C.E., Da Silva, A.S., Almeida, T.C., Hermes, C.L., Wolkmer, P.,
338
Silva, C.B., Moreira, K.L.S., Beck, R.C.R., Moresco, R.N., Da Veiga, M.L., Stefani,
339
L.M., Monteiro, S.G., 2014. Trypanocidal activity of free and nanoencapsulation
340
curcumin on Trypanosoma evansi. Parasitol. 142, 439-448.
M AN U
SC
336
341
Joshi, P.P., Shegokar, V.R., Powar, R.M., Herder, S., Katti, R., Salkar, H.S., Dani, V.S.,
343
Bhargava, A., Jannin, J., Truc, P., 2005. Human trypanosomosis caused by
344
Trypanosoma evansi in India: the first case report. Am. J. Trop. Med. Hyg. 73, 491-495.
345
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342
Lopes, N.P., Kato, M.J., Andrade, E.H., Maia, J.G., Yoshida, M., Planchart, A.R.,
347
Katzin, A.M., 1999. Antimalarial use of volatile oil from leaves of Vitola surinamensis
348
(Rol.) Warb. By Waiapi Amazon Indians. J. Ethnopharmacol. 67, 313-319.
349
EP
346
Masocha W., Rottenberg M.E., Kristensson K., 2007. Migration of African
351
trypanosomes across the blood-brain barrier. Physiol. Behav 92, 110-114.
352
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353
Medeiros, M.G.F., da Silva, A.C., Citó, A.M.G.L., Borges, A.R., de Lima, S.G., Lopes,
354
J.A.D., Figueiredo, B.Q., 2011. In vitro antileishmanial activity and Cytotoxicity of
355
essential oil from Lippia sidoides Cham. Parasitol. Int. 60, 237-241.
356 357
Morales-Yuste, M., Morrilas-Márquez, F., Martín-Sanchez, J., Valero-López, A.,
358
Navarro-Moll, M.C., 2010. Activity of (-) α-bisabolol against Leishmania infantum
359
promastigotes. Phytomedicine 17, 279-281.
360
ACCEPTED MANUSCRIPT Morilla, M.J., Romero, E.L., 2015. Nanomedicine against Chagas disease: an update on
362
therapeuthics, prophylaxis and diagnosis. Nanomedicine 10, 465-481.
363
Mosqueira, V.C.F., Loiseau, P.M., Bories, C., Legrand, P., Devissaguet, J.P., Barrat, G.,
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2004. Efficacy and pharmacokinetics of intravenous nanocapsules formulations of
365
halofantrine in Plasmodium berghei-infected mice. Antimicrob. Agents Chemother 48,
366
1222-1228.
RI PT
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Nayak, A.P., Tiyaboonchai, W., Patankar, S., Madhusudhan, B., Souto, E.B., 2010.
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Curcuminoids-loaded lipid nanoparticles: Novel approach towards malaria treatment.
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Colloids Surf. B: Biointerfaces 81, 263-273.
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Raffin, R.P., Lima, A., Lorenzoni, R., Antonow, M.B., Turra, C., Alves, M.P., Fagan,
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S.B.,
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characterization and in vivo antiedematogenic and antinociceptive activities. J. Biomed.
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Nanotechnol. 8, 309-315, 2012.
Natural
lipid
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nanoparticles
containing
M AN U
2012.
nimesulide:
synthesis,
Rottini, M.M,. Amaral, A.C.F, Ferreira, J.L.P., Silva, J.R.A., Taniwaki, N.N., de Souza,
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C.S.F., d’Escoffier, L.N., Almeida-Souza, F., Hardoim, D.J., da Costa, S.C.G.,
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Calabrese, K.S., 2015. In vitro evaluation of (-) α-bisabolol as a promising agent against
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Leishmania amazonensis. Exp. Parasitol. 148, 66-72.
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Salomão, K., De Santana, N.A., Molina, M.T., De Castro, S.L., Menna-Barreto, R.F.S.,
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2013. Trypanosoma cruzi mitochondrial swelling and membrane potential colapse as
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primary evidence of the mode of action naphtoquinone analogues. BMC Microbiol. 3,
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1-12.
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Silva, R.A.M., Seidl A., Ramirez L., Dávila A.M.R., 2002. Trypanosoma evansi e
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Trypanosoma vivax: Biologia, Diagnóstico e controle, Corumbá, Embrapa Pantanal.
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Souto, E.B., 2009. A special issue on lipid-based delivery systems (liposomes, lipid
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nanoparticles, lipid matrices and medicines). J. Biomed. Nanotechnol. 5, 315-316.
392
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Souto, E.B., Muller, R.H., 2010. Lipid nanoparticles: effect on bioavailability and
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pharmacokinetic and biopharmaceutical changes. Handb. Exp. Pharmacol. 197, 115-
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141, 2010.
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Vendrametto, M.C., Santos, A.O., Nakamura, C.V., Dias Filho, B.P., Cortez, D.A.,
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Ueda-Nakamura, T., 2010. Evaluation of antileishmanial activity of eupomatenoid-5, a
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compound isolated from leaves of Piper regnellii var. pallescens. Parasitol. Int. 59, 154-
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158.
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Watkins, R., Wu, L., Zhang, C., Davis, R.M., Xu, B., 2015. Natural product-based
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nanomedicine: recent advances and issues. Int. J. Nanomedicine 10, 6055-6074.
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Table 1: Composition of solid lipid nanoparticles containing α-bisabolol (SLN-B). Compounds Lipid phase
%
Shea butter
5.0
2.0
SC
Sorbitan monooleate
0.3
Polyssorbate 80 Milli-Q water
436
437
438
439
440
441
442
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α-bisabolol Aqueous phase
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% 3.0 89.7
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Table 2 - Physicochemical properties of solid lipid nanoparticles containing α-bisabolol (SLN-B). Suspension SLN-B
Polydispersity index
Z- Potential (mV)
pH
191.8 ± 1.13
0.317 ±0.0011
-7.76 ± 1.13
6.83
Note: Mean ± standard deviation of three determinations of a same formulation.
SC
448 449
Size (nm)
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451
452
457
458
459
460
461
462
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454
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Table 3: Mean and standard deviation of the prepatent period, longevity, mortality and therapeutic
465
success using the treatments with α-bisabolol and solid lipid nanoparticles (SLN-B), and diminazene
466
aceturate (D.A.) in mice experimentally infected by T. evansi. Groups (n=6)
Treatment
Prepatent period (Day)
A
Negative control
-
B
Positive control
1.0c (± 0.0)
C
Bisabolol (1.0 mL kg-1)
1.0c (± 0.0)
D
SLN-B (1.0 mL kg-1)
E
D.A. (3.5 mg Kg-1)
F
D.A. (3.5 mg Kg-1) + SLN-B (1.0 mL kg-1)
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Longevity (Day)
Mortality (n)
Therapeutic success (%)
0/6
-
4.5c (±0.5)
6/6
0
5.0c (±0.5)
6/6
0
1.3c (± 0.7)
32.3b (±7.6)
5/6
16.66
14.1b (± 2.2)
30.2b (±3.0)
5/6
16.66
34.1a (± 5.0)
48.2a (±3.0)
3/6
50
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60.0a (±0.0)
Means followed by the same letter in the same columns do not differ significantly according to the
468
Bonferroni post-hoc test. The experiment lasted 60 days after infection.
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Figure 1: In vitro activity of different concentrations of bisabolol [A] and solid lipid
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nanoparticles containing bisabolol (SLN-B) [B] against trypomastigotes forms of
484
Trypanosoma evansi. Results within a circle were not statistically different (p>0.05), at
485
the same time (h), using the Bonferroni post-hoc test.
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ACCEPTED MANUSCRIPT Trypanosoma evansi has been recorded several cases of resistance to antiprotozoal drugs Alpha-Bisabolol and SLN-B have trypanocidal action in vitro
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Association of SLN-B and diminazene aceturate potentiates the curative effect of T. evansi in mice
S0014-4894(16)30213-2
DOI:
10.1016/j.exppara.2016.09.018
Reference:
YEXPR 7309
To appear in:
Experimental Parasitology
Received Date: 24 July 2016 Revised Date:
15 September 2016
Accepted Date: 27 September 2016
Please cite this article as: Baldissera, M.D., Grando, T.H., de Souza, C.F., Cossetin, L.F., da Silva, A.P.T., Giongo, J.L., Monteiro, S.G., A nanotechnology based new approach for Trypanosoma evansi chemotherapy: In vitro and vivo trypanocidal effect of (-)-α-bisabolol, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.09.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A nanotechnology based new approach for Trypanosoma evansi chemotherapy: in
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vitro and vivo trypanocidal effect of (-)-α-Bisabolol
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Matheus D. Baldisseraa*, Thirssa H. Grandoa, Carine F. de Souzab, Luciana F. Cossetina,
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Ana P.T. da Silvac, Janince L. Giongod, Silvia G. Monteiroa*.
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a
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(UFSM), Santa Maria, RS, Brazil.
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b
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Department of Microbiology and Parasitology, Universidade Federal de Santa Maria
Department of Physiology and Pharmacology, Universidade Federal de Santa Maria
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(UFSM), Santa Maria, RS, Brazil.
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c
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Brazil
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d
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Uruguai e das Missões/URI, Santiago, RS, Brazil.
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Laboratory of Pharmaceutical Technology, Universidade Regional Integrada do Alto
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Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, RS,
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*Author for correspondence: [email protected] (M.D. Baldissera) and
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[email protected] (S.G. Monteiro).
ACCEPTED MANUSCRIPT ABSTRACT
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The aim of this study was to evaluate the in vitro and in vivo susceptibility of
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Trypanosoma evansi to α-Bisabolol and solid lipid nanoparticles containing α-Bisabolol
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(SLN-B). In vitro, a trypanocidal effect of α-Bisabolol and SLN-B was observed when
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used at 0.5, 1 and 2 % concentrations, i.e., the concentrations of 1 and 2 % showed a
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faster trypanocidal effect when compared to chemotherapy (diminazene aceturate –
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D.A.). T. evansi infected mice were treated with α-Bisabolol and SLN-B at a dose of 1.0
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mL kg-1 during seven days via oral gavage. In vivo, treatment with SLN-B, D.A. and
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D.A. associated with SLN-B were able to increase (p<0.05) the pre-patent period and
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longevity when compared to positive control (infected and untreated animals), but
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showed no curative efficacy. T. evansi infected mice treated with D.A. associate with
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SLN-B, where a curative efficacy of 50 % was found, a much better result when D.A
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and SLN-B were used alone (16.66 %). In summary, the association with D.A + SLN-B
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can be used as an alternative to improve the therapeutic effectiveness of D.A., and for
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treatment of infected animals with T. evansi. Also, the nanotechnology associated with
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natural products arises an important alternative for the improve the trypanocidal action.
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Keywords: “surra”; nanotechnology; alternative treatment.
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1 INTRODUCTION
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Trypanosoma evansi is the most widely distributed of the pathogenic African
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trypanosomes, affecting domestic and wild animals (Silva et al., 2002; Desquesnes et
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al., 2013), but rarely parasitizes humans (Joshi et al., 2005). The parasite causes a
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disease known as “surra”, and it is mechanically transmitted by biting flies (Stomoxis
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sp., Hematopota sp., and Chrysops sp.), and the principal host species varies according
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to geographical location, parasitizing principally horses, camels, cattle, dogs and cats
ACCEPTED MANUSCRIPT (Desquesnes et al., 2013). Diminazene aceturate (D.A.) at a single dose of 3.5 mg kg-1 is
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capable to providing an elimination of parasites in bloodstream a few hours after
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administration, however, it has no curative efficacy because relapses parasitemia
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occurring due trypanosomes pass through the blood-brain barrier, finding allocated in
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central nervous system, a refuge area for T. evansi during the residual period of the drug
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circulation. Diminazene aceturate does not cross the blood-brain barrier in amounts
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sufficient to eliminate all the parasites (Masoha et al., 2007). Also, therapeutic dose of
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D.A. usually shows signs of toxicity, especially related to hepatotoxic and nephrotoxic
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effects, as reported in the literature (Baldissera et al., 2016a; Baldissera et al., 2016b).
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α-Bisabolol ((-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)5-heptein-2-ol)), also
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known as levomenol, is an sesquiterpene alcohol present in essential oils of several
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plants. With respect to the antiparasitic effect of α-bisabolol, it has shown strong
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antileishmanial activity (Morales-Yuste et al., 2010; Rottini et al., 2015, Corpas-López
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et al., 2015). Regarding the leishmanicidal activity, α-bisabolol has been considered a
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promising compound for the treatment of visceral leishmaniasis, that was able to
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eliminate the promastigotes forms of Leishmania infantum and Leishmania amazonensis
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(Rottini et al., 2015).
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Nanomedicine have been studied for treatment of parasite infections because
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they are able to deliver the drug to the specific target in human and animals body where
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the parasite is located, such as blood and tissues (Mosqueira et al., 2004; Branquinho et
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al., 2014; Morilla and Romero, 2015). Solid lipid nanoparticles (SLN) are a specific
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type of nanoparticle that have been used successfully because protect the drugs from
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chemical degradation, possibility of sustained drug release and enhanced drug
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solubility, (Souto 2009; Souto and Muller 2010). Different antiparasitic drug have been
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loaded into SLN for improving the therapeutic efficacy, such as curcuminoids for
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malaria treatment (Nayak et al., 2010). According to Watkins et al. (2015) the utilization of nanotechnology with
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natural compounds is a rapidly developing field due advantages to the delivery in the
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treatment of parasites diseases. The incorporation of nanoparticles can increase the
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bioavailability, targeting, and controlled-release profiles of the natural products.
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Example of this is the encapsulation of lactone, a natural compound, against Chagas
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disease (Branquinho et al., 2014). Against T. evansi infection, natural products
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encapsulated has established promising results such use of curcumin (Gresller et al.,
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2015), and Melaleuca alternifolia and Achyrocline satureioides essential oils
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(Baldissera et al., 2014, Do Carmo et al., 2015), when compared to the free form.
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Based in this evidences, the aims of this study was to develop and characterize
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solid lipid nanoparticles containing α-bisabolol (SLN-B), and to evaluate their efficacy
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in vitro and in vivo against T. evansi.
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2 MATERIALS AND METHODS
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2.1. (-)-α-Bisabolol
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2.2. Preparation of SLN-B
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(-)-α-Bisabolol (molecular weight: 222.37 g/mol) was purchased from Sigma-
Aldrich® and exhibit 93 % of purity.
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The suspension of SLN-B was prepared according the method described by
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Raffin et al. (2012). The components of the SLN-B are described in Table 1. The
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components of the lipid phase and aqueous phase were separately placed in a beaker and
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and the active principle (AP) was added to the lipid phase at the end of the heating
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period. With the aid of a funnel, the aqueous phase was poured into the lipid phase and
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stirring is continued for 10 minutes. The dispersion of nanoparticles was performed
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using the Ultra-Turrax® T25 during 20 min at 20.000 rpm.
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2.3. Characterization of SLN-B
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The pH determination was performed directly in the suspensions in
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potentiometer (Digimed®) previously calibrated with buffer solutions pH 4.0 and 7.0.
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The determination of the diameter and polydispersity of nanoparticles in suspension
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were performed by dynamic light scattering, the Zetasizer®, Nano-ZS from Malvern
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equipment. The suspensions were diluted 500 times (v:v) in Milli-Q water and the
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results were determined by the average of three replicates. The zeta potential of the
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nanospheres suspensions were obtained using the technique of electrophoretic mobility
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Zetasizer®, Malvern Nano-ZS instrument. The samples were pre-diluted at 500 times
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(v:v) in 10 mM sodium chloride and filtered through a membrane with 0.45
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micrometers. The results are expressed in millivolts (mV) from a mean of three
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determinations.
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2.4. Trypanosoma evansi isolate This study was conducted in two consecutive experiments (in vitro and in vivo).
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The cryopreserved isolate of T. evansi used in these experiments was obtained from a
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naturally infected dog (Colpo et al. 2005). Two rats (Ra and Rb) were infected
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intraperitoneally with blood contaminated by trypomastigotes, which was kept
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cryopreserved in liquid nitrogen. This process was performed in order to obtain a large
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amount of viable parasites for in vitro tests (Ra), and to infect the mice in the
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experimental groups (Rb).
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2.5. In vitro test The culture medium for T. evansi was adapted from Baltz et al. (1985) as
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previously published by Baldissera et al. (2013). The protozoans were acquired from the
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rat infected with the T. evansi isolate (Ra). At three days post-infection, the rat showed
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high parasitemia (3.0 x 104 trypanosomes/µL) and it was anesthetized with isoflurane
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for blood collection by cardiac puncture. The blood was stored in tubes containing
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EDTA (ethylenediamine tetraacetic acid). For trypanosomes separation, each 200 µL of
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blood was diluted in complete culture medium (DMEM) 1:1 (v/v), added to
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microcentrifuge tubes and centrifuged at 400 x g for 10 minutes at 25 °C. The
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supernatant was removed and resuspended in culture medium and the number of
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parasites was counted in a Neubauer chamber.
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The culture medium containing the parasites was distributed in microtiter plates
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(270 µL/well), and 25 µL of each treatment were added (diluted in culture medium). For
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this assessment, the α-bisabolol and SLN-B were used at concentrations of 0.5, 1.0 and
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2.0%. A positive control (0.5% D.A.) was also adopted at the same volume (25 µL). 20
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µL of each well was used to the count the number of parasites at 1, 3, 6 and 9 hours
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after the onset of the experiment in the Neubauer chambers. The in vitro tests were
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performed in duplicate.
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2.6. In vivo test
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2.6.1. Animal model
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weighing an average of 28 ± 0.5 g were used as the experimental model. The animals
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were maintained under controlled light and environment (12:12 h light-dark cycle, 22 ±
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1 °C, 70% relative humidity) with free access to commercial food and water.
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Experiments were carried out between 8 am and 5 pm. All animals were subjected to a
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period of 15 days for adaptation. All efforts were made to minimize animal suffering
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and to reduce the number of animals used in the experiments.
2.6.2. Experimental design and parasitemia estimation
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The mice were assigned to six groups (A - F), with six animals each. Group A
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consisted of uninfected and untreated animals (negative control); Group B: infected and
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untreated mice (positive control); Group C: animals infected and treated α-bisabolol 1.0
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mL kg-1; Group D: animals infected and treated with SLN-B 1.0 mL kg-1; Group E:
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animals infected and treated with D.A; Group F: infected and treated with combination
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of D.A. (3.5 mg kg-1 – intramuscularly) and SLN-B (1.0 mL kg-1 – orally). The infected
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animal groups were inoculated intraperitoneally with 0.05 mL of blood from Rb
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containing 2.0 x 105 trypanosomes.
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The D.A. was intramuscularly injected in a single dose of 3.5 mg kg-1, according
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the manufactures recommendation. At one hour post-infection, α-bisabolol and SLN-B
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were orally administered. A daily supply of α-bisabolol and SLN-B was maintained for
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seven days according the protocol used by Corpas-López et al. (2015).
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The peripheral blood from the tail of the rats was examined daily for scoring the
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degree of parasitemia. Each slide was prepared with fresh blood collected from the tail
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coccygeal vein, stained by the Romanowski method, and visualized at a magnification
ACCEPTED MANUSCRIPT 167
of 1,000 x according to the method described by Da Silva et al. (2006). The mice were
168
observed for up to 60 days.
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2.6.3. Treatment efficacy The number of mice that did not show clinical signs of T. evansi infection and
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did not die after treatment determined the treatment efficacy. Prepatent period,
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longevity and animal mortality were also observed.
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The data met the assumption of parametric testing according to the Kolmogorov-
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Smirnov test. The bilateral two-way analysis of variance (ANOVA) followed by the
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Bonferroni post-hoc test were used for comparison of means. Differences between
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groups were rated significant at p<0.05. All analysis were carried out in an IBM
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compatible computer using the Statistical Package for the Social Sciences (SPSS)
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software 20. Results were presented as means ± standard deviation.
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3 RESULTS
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3.1. Characterization of SLN-B
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SLN-B was evaluated regarding their physical and chemical properties (Table
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2). The particle size was around 191.8 nanometers, and the polydispersion index was
188
0.317, with a zeta potential of – 7.76 mV and pH 6.83.
189 190
3.2. In vitro test
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192
proportional to the concentration used (Figure 1). A reduction of live trypomastigotes
193
was observed at all concentrations when compared to the control group. After 6h, there
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were no living trypomastigotes in 1 % and 2 % concentrations using α-bisabolol. After
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9 h of assay, there were also no living trypomastigotes in 0.5 % concentration as well as
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on the D.A. treatment (Figure 1-A)
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The SLN-B are shown in Figure 1-B. Similarly, a reduction of live
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trypomastigotes was observed at all concentrations when compared to the control group.
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After 6h, there were no living trypomastigotes in 1 % and 2 % concentrations. After 9 h
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of assay, there were also no living trypomastigotes in 0.5 % concentration as well as on
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the D.A. treatment. A contrary, in control samples the parasites were all alive, what
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validates our experiment.
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3.3. In vivo test
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Longevity of the group A exactly represented by the days that the experiment
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lasted (60 days) (Table 3). Longevity in the groups B, C, D, E and F were 4.5, 5.0, 32.3,
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30.2 and 48.2 days, respectively. The groups D (SLN-B), E (D.A.) and F (D.A + SLN-
208
B) had no curative efficacy, but increased longevity compared to the group B (positive
209
control) (p<0.05). The group F showed an increase (p<0.05) longevity when compared
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the treatment with SLN-B and D.A. isolated, and showed 50 % of curative
211
effectiveness, while the groups D and E showed 16.66 % of therapeutic effectiveness.
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Prepatent period increased in the group D, E and F when compared to group B (p<0.05),
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but the prepatent period is higher in the group F compared to groups D and E.
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4. DISCUSSION
ACCEPTED MANUSCRIPT Natural products and their compounds are some of the most interesting sources
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of new drugs, since the chemotherapy of T. evansi infection present serious limitations.
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According Tamma et al. (2012) combination therapy is considered as a new and
219
promising tool because enhance their parasitic performance and decrease development
220
of drug resistance via synergistic or agonistic interactions between used drugs, such as
221
observed in this study. In this sense, a combination of D.A. and SLN-B exhibited
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superior activity against T. evansi when compared to the use of D.A. individual.
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On the in vitro assessment, the trypanocidal activity against T. evansi was
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verified to be proportional to the dose applied for α-bisabolol and SLN-B formulations.
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Previous studies suggest that α-bisabolol shows antiparasitic activity in vitro against
226
other important blood protozoa, such as L. amazonensis and L. infantum. Recent study
227
demonstrated that α-bisabolol showed significant antileishmanial activity against
228
promastigotes with a 50 % effective concentration (IC50) of 8.07 µg/mL (24 h) and 4.26
229
µg/mL (48 h). Against intracellular amastigotes the IC50 of α-bisabolol was 4.15 µg/mL
230
(Rottini et al., 2015). Also, Morales-Yust et al. (2015) demonstrated that 1000 and 500
231
µg/mL of α-bisabolol achieved 100 % inhibition of promastigote form of L. infantum in
232
vitro. According to recent studies, the alterations in membrane permeability, principally
233
in the mitochondrial membrane, may lead to parasite death (Salomão et al., 2013).
234
According this author, the possible mechanism action of α-bisabolol is related with the
235
capacity to cross the mitochondrial membrane, which can lead to parasite death. Also,
236
studies have been demonstrated that sesquiterpenes compounds are capable to inhibit
237
the synthesis of ergosterol, an important component of cell membrane. This fact has
238
been reported by many authors attempting to explain the antileishmanial action of
239
sesquiterpenes compounds extracted from plants (Vendramentto et al., 2010; Medeiros
240
et al., 2011; Baldissera et al., 2016c).
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ACCEPTED MANUSCRIPT Based on these previous promising in vitro results, we have designed an in vivo
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experiment using mice infected by T. evansi as the experimental model Although the
243
therapeutic protocol used with α-bisabolol and SLN-B did not present curative efficacy,
244
an increased in longevity of animals treated with SLN-B was observed. In addition,
245
SLN-B showed a similar curative efficacy when compared to D.A. It is important
246
emphasize that nanotechnology was able to ameliorate the trypanocidal action of α-
247
bisabolol, due the increase of longevity and the presence of efficacy therapeutic (16.66
248
%) when compared to free α-bisabolol that showed no curative effectiveness (0%),
249
similar results observed by Gressler et al. (2015) using curcumin and Baldissera et al.
250
(2016c) using α-terpinene against T. evansi. Recently, study published by Watkins et al.
251
(2015) demonstrated that natural product-based on nanotechnology would be a major
252
advance in the efforts to increase their therapeutic effects. The main features of SLN are
253
physical
254
biocompatibility and degradability (Nayak et al., 2010).
255
nanoparticles can significantly increase the bioavaibility of natural products in vivo, due
256
this capability of manipulate the particles in order to target specific areas of the body
257
and control the release of drugs, similar observed by Nayak et al. (2010) using
258
curcuminoids against Plasmodium berghei.
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excipients
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Thus, we believe that
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In addition, the association of D.A. with SLN-B increased longevity compared
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to the group B (positive control), and had 50 % of therapeutic efficacy, compared to
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group C (treated only D.A.), with 16.66 % of therapeutic efficacy. Therefore, the D.A or
262
SLN-B alone would not be successful in the treatment of trypanosomosis, but when
263
using associated, a synergic effect occurs, which could result on a new option for this
264
disease treatment. Recently, a study demonstrated that D.A., when associated with other
ACCEPTED MANUSCRIPT 265
terpene compound (α-terpinene), showed an increase of therapeutic efficacy (57.14 %)
266
when compared to D.A. alone (14.28 %). α-Bisabolol, in its conventional and nanostructured forms has a trypanocidal
268
action in vitro, but only nanostructured leads to higher animals longevity, but it was not
269
effective in the treatment of mice experimentally with T. evansi. D.A. when associated
270
with SLN-B achieved therapeutic success, becoming an alternative method to treat and
271
control infections caused by T. evansi.
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Ethics Committee
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The procedures were approved by the Animal Welfare Committee of Federal University
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of Santa Maria under number 6583091215.
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REFERENCES
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Baldissera, M.D., Da Silva, A.S., Oliveira, C.B., Zimmermann, C.E.P., Vaucher, R.A.,
279
Santos, R.C.V., Rech, V.C., Tonin, A.A., Giongo, J.L., Mattos, C.B., Koester, L.,
280
Santurio, J.M., Monteiro, S.G., 2013. Trypanocidal activity of the essential oils in their
281
conventional and nanoemulsion forms: In vitro tests. Exp. Parasitol.134, 356-361.
EP
282
TE D
277
Baldissera, M.D., Oliveira, C.B., Rech, V.C., Rezer, J.F.P., Sagrillo, M.R., Alves, M.P.,
284
da Silva, A.P.T., Leal, D.B.R., Boligon, A.A., Athayde, M.L., Da Silva, A.S., Mendes,
285
R.E., Monteiro, S.G., 2014. Treatment with essential oil of Achyrocline satureioides in
286
rats infected with Trypanosoma evansi: Relationship between protective effect and
287
tissue damage. Pathol. Res. Pract. 210, 1068-1074.
AC C
283
288 289
Baldissera, M.D., Bottari, N.B., Rech, V.C., Nishihira, V.S.K., Oliveira, C.B., Cargnin,
290
L.P., Moresco, R.N., Thomé, G.R., Schetinger, M.R.C., Morsch, V.M., Monteiro, S.G.,
291
Tonin, A.A., Da Silva, A.S., 2016a. Combination of diminazene aceturate and
292
resveratrol reduces the toxic effects of chemotherapy in treating Trypanosoma evansi
293
infection. Comp. Clin. Pathol. 25, 137-144.
ACCEPTED MANUSCRIPT 294
Baldissera, M.D., Gonçalves, R.A., Sagrillo, M.R., Grando, T.H., Ritter, C.S., Grotto,
296
F.S., Brum, G.F., da Luz, S.C.A., Silveira, S.O., Fausto, V.P., Boligon, A.A., Vaucher,
297
R.A., Stefani, L.M., da Silva, A.S., Souza, C.F., Monteiro, S.G., 2016b. Effects of
298
Treatment with the anti-parasitic drug diminazene aceturate on antioxidant enzymes in
299
rat liver and kidney. Naunyn-Schmiedeberg’s Arch. Pharmacol. 389, 429-438.
RI PT
295
300
Baldissera, M.D., Grando, T.H., Souza, C.F., Gressler, L.T., Stefani, L.M., Da Silva,
302
A.S., Monteiro, S.G., 2016c. In vitro and in vivo action of α-terpinen-4-ol, γ-terpinene
303
and α-terpinene against Trypanosoma evansi. Exp. Parasitol. 162, 43-48.
SC
301
304
Baltz, T., Baltz, D., Giroud, C., Crockett, J., 1985. Cultivation in a semi-defined
306
medium of animals infective forms of Trypanosoma brucei, T. equiperdum, T. evansi,
307
T. rhodesiensi and T. gambiense. The EMBO Journal 4, 1273-1277.
M AN U
305
308
Branquinho, R.T., Mosqueira, V.C.F., de Oliveira-Silva, J.C.V., Simões-Silva, M.R.,
310
Saúde-Guimarães, D.A., de Lana, M., 2014. Sesquiterpene Lactone in Nanostructured
311
parenteral dosage form is efficacious in experimental Chagas disease. Antimicrob.
312
Agents Chemother. 58, 2067-2075.
TE D
309
313
Colpo, C.B., Monteiro, S.G., Stainki, D.R., Colpo, E.T.B., Henriques, G.B., 2005.
315
Natural infection by Trypanosoma evansi in dogs. Cienc. Rural 35, 717-719.
316
EP
314
Corpas-López, V., Morillas-Márquez, F., Navarro-Moll, M.C., Merino-Espinosa, G.,
318
Díaz-Sáez, V., Martín-Sánchez, J., 2015. (-) α-bisabolol, a promising oral compound for
319
the Treatment of visceral leishmaniasis. J. Nat. Prod. 78, 1202-1207.
320
AC C
317
321
Da Silva, A.S., Doyle, R.L., Monteiro, S.G., 2006. Métodos de contenção e confecção
322
de esfregaço sanguíneo para pesquisa de hemoparasitas em ratos e camundongos. Rev.
323
FZVA 13, 83-87.
324 325
Desquesnes, M., Holzmuller, P., Lai, H., Dargantes, A., Lun, Z.L., Jittaplapong, S.,
326
2013. Trypanosoma evansi and Surra: A review and perspectives on origin, history,
ACCEPTED MANUSCRIPT distribution, taxonomy, morphology, hosts and pathogenic effects. BioMed Res. Int.
328
2013, 19416.
329
Do Carmo, G.M., Baldissera, M.D., Vaucher, R.A., Rech, V.C, Oliveira, C.B., Sagrillo,
330
M.R., Boligon, A.A., Athayde, M.L., Alves, M.P., França, R.T., Lopes, S.T.A.,
331
Schwertz, C.I., Mendes, R.E., Monteiro, S.G., Da Silva, A.S., 2015. Effect of the
332
treatment with Achyrocline satureioides (free and nanocapsules essential oil) and
333
diminazene aceturate on hematological and biochemical parameters in rats infected by
334
Trypanosoma evansi. Exp. Parasitol. 149, 39-46
RI PT
327
335
Gressler, L.T., Oliveira, C.B., Coradini, K., Dalla Rosa, L., Grando, T.H., Baldissera,
337
M.D., Zimmermann, C.E., Da Silva, A.S., Almeida, T.C., Hermes, C.L., Wolkmer, P.,
338
Silva, C.B., Moreira, K.L.S., Beck, R.C.R., Moresco, R.N., Da Veiga, M.L., Stefani,
339
L.M., Monteiro, S.G., 2014. Trypanocidal activity of free and nanoencapsulation
340
curcumin on Trypanosoma evansi. Parasitol. 142, 439-448.
M AN U
SC
336
341
Joshi, P.P., Shegokar, V.R., Powar, R.M., Herder, S., Katti, R., Salkar, H.S., Dani, V.S.,
343
Bhargava, A., Jannin, J., Truc, P., 2005. Human trypanosomosis caused by
344
Trypanosoma evansi in India: the first case report. Am. J. Trop. Med. Hyg. 73, 491-495.
345
TE D
342
Lopes, N.P., Kato, M.J., Andrade, E.H., Maia, J.G., Yoshida, M., Planchart, A.R.,
347
Katzin, A.M., 1999. Antimalarial use of volatile oil from leaves of Vitola surinamensis
348
(Rol.) Warb. By Waiapi Amazon Indians. J. Ethnopharmacol. 67, 313-319.
349
EP
346
Masocha W., Rottenberg M.E., Kristensson K., 2007. Migration of African
351
trypanosomes across the blood-brain barrier. Physiol. Behav 92, 110-114.
352
AC C
350
353
Medeiros, M.G.F., da Silva, A.C., Citó, A.M.G.L., Borges, A.R., de Lima, S.G., Lopes,
354
J.A.D., Figueiredo, B.Q., 2011. In vitro antileishmanial activity and Cytotoxicity of
355
essential oil from Lippia sidoides Cham. Parasitol. Int. 60, 237-241.
356 357
Morales-Yuste, M., Morrilas-Márquez, F., Martín-Sanchez, J., Valero-López, A.,
358
Navarro-Moll, M.C., 2010. Activity of (-) α-bisabolol against Leishmania infantum
359
promastigotes. Phytomedicine 17, 279-281.
360
ACCEPTED MANUSCRIPT Morilla, M.J., Romero, E.L., 2015. Nanomedicine against Chagas disease: an update on
362
therapeuthics, prophylaxis and diagnosis. Nanomedicine 10, 465-481.
363
Mosqueira, V.C.F., Loiseau, P.M., Bories, C., Legrand, P., Devissaguet, J.P., Barrat, G.,
364
2004. Efficacy and pharmacokinetics of intravenous nanocapsules formulations of
365
halofantrine in Plasmodium berghei-infected mice. Antimicrob. Agents Chemother 48,
366
1222-1228.
RI PT
361
367
Nayak, A.P., Tiyaboonchai, W., Patankar, S., Madhusudhan, B., Souto, E.B., 2010.
369
Curcuminoids-loaded lipid nanoparticles: Novel approach towards malaria treatment.
370
Colloids Surf. B: Biointerfaces 81, 263-273.
371
SC
368
372
Raffin, R.P., Lima, A., Lorenzoni, R., Antonow, M.B., Turra, C., Alves, M.P., Fagan,
373
S.B.,
374
characterization and in vivo antiedematogenic and antinociceptive activities. J. Biomed.
375
Nanotechnol. 8, 309-315, 2012.
Natural
lipid
376
nanoparticles
containing
M AN U
2012.
nimesulide:
synthesis,
Rottini, M.M,. Amaral, A.C.F, Ferreira, J.L.P., Silva, J.R.A., Taniwaki, N.N., de Souza,
378
C.S.F., d’Escoffier, L.N., Almeida-Souza, F., Hardoim, D.J., da Costa, S.C.G.,
379
Calabrese, K.S., 2015. In vitro evaluation of (-) α-bisabolol as a promising agent against
380
Leishmania amazonensis. Exp. Parasitol. 148, 66-72.
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377
381
Salomão, K., De Santana, N.A., Molina, M.T., De Castro, S.L., Menna-Barreto, R.F.S.,
383
2013. Trypanosoma cruzi mitochondrial swelling and membrane potential colapse as
384
primary evidence of the mode of action naphtoquinone analogues. BMC Microbiol. 3,
385
1-12.
AC C
386
EP
382
387
Silva, R.A.M., Seidl A., Ramirez L., Dávila A.M.R., 2002. Trypanosoma evansi e
388
Trypanosoma vivax: Biologia, Diagnóstico e controle, Corumbá, Embrapa Pantanal.
389 390
Souto, E.B., 2009. A special issue on lipid-based delivery systems (liposomes, lipid
391
nanoparticles, lipid matrices and medicines). J. Biomed. Nanotechnol. 5, 315-316.
392
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Souto, E.B., Muller, R.H., 2010. Lipid nanoparticles: effect on bioavailability and
394
pharmacokinetic and biopharmaceutical changes. Handb. Exp. Pharmacol. 197, 115-
395
141, 2010.
396
Vendrametto, M.C., Santos, A.O., Nakamura, C.V., Dias Filho, B.P., Cortez, D.A.,
398
Ueda-Nakamura, T., 2010. Evaluation of antileishmanial activity of eupomatenoid-5, a
399
compound isolated from leaves of Piper regnellii var. pallescens. Parasitol. Int. 59, 154-
400
158.
RI PT
397
401
Watkins, R., Wu, L., Zhang, C., Davis, R.M., Xu, B., 2015. Natural product-based
403
nanomedicine: recent advances and issues. Int. J. Nanomedicine 10, 6055-6074.
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Table 1: Composition of solid lipid nanoparticles containing α-bisabolol (SLN-B). Compounds Lipid phase
%
Shea butter
5.0
2.0
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Sorbitan monooleate
0.3
Polyssorbate 80 Milli-Q water
436
437
438
439
440
441
442
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433
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α-bisabolol Aqueous phase
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431 432
% 3.0 89.7
ACCEPTED MANUSCRIPT 443
444
Table 2 - Physicochemical properties of solid lipid nanoparticles containing α-bisabolol (SLN-B). Suspension SLN-B
Polydispersity index
Z- Potential (mV)
pH
191.8 ± 1.13
0.317 ±0.0011
-7.76 ± 1.13
6.83
Note: Mean ± standard deviation of three determinations of a same formulation.
SC
448 449
Size (nm)
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450
451
452
457
458
459
460
461
462
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455
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453
454
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445 446 447
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Table 3: Mean and standard deviation of the prepatent period, longevity, mortality and therapeutic
465
success using the treatments with α-bisabolol and solid lipid nanoparticles (SLN-B), and diminazene
466
aceturate (D.A.) in mice experimentally infected by T. evansi. Groups (n=6)
Treatment
Prepatent period (Day)
A
Negative control
-
B
Positive control
1.0c (± 0.0)
C
Bisabolol (1.0 mL kg-1)
1.0c (± 0.0)
D
SLN-B (1.0 mL kg-1)
E
D.A. (3.5 mg Kg-1)
F
D.A. (3.5 mg Kg-1) + SLN-B (1.0 mL kg-1)
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Longevity (Day)
Mortality (n)
Therapeutic success (%)
0/6
-
4.5c (±0.5)
6/6
0
5.0c (±0.5)
6/6
0
1.3c (± 0.7)
32.3b (±7.6)
5/6
16.66
14.1b (± 2.2)
30.2b (±3.0)
5/6
16.66
34.1a (± 5.0)
48.2a (±3.0)
3/6
50
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60.0a (±0.0)
Means followed by the same letter in the same columns do not differ significantly according to the
468
Bonferroni post-hoc test. The experiment lasted 60 days after infection.
471 472 473 474 475 476 477 478 479
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Figure 1: In vitro activity of different concentrations of bisabolol [A] and solid lipid
483
nanoparticles containing bisabolol (SLN-B) [B] against trypomastigotes forms of
484
Trypanosoma evansi. Results within a circle were not statistically different (p>0.05), at
485
the same time (h), using the Bonferroni post-hoc test.
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ACCEPTED MANUSCRIPT Trypanosoma evansi has been recorded several cases of resistance to antiprotozoal drugs Alpha-Bisabolol and SLN-B have trypanocidal action in vitro
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Association of SLN-B and diminazene aceturate potentiates the curative effect of T. evansi in mice